Compositions and method for rapid, real-time detection of influenza a virus (H1n1) swine 2009 (14-Jan-2010)

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patent application Ser. No. 11/844,933, filed Aug. 24, 2007, now pending; and claims the benefit of U.S. Provisional Application No. 60/843,711, filed Sep. 12, 2006, now expired, the entire contents of each of which is specifically incorporated herein in its entirety by express reference thereto.

FIELD OF THE INVENTION

The invention relates to a biological organism detection product and methods of using the same to: 1) rapidly identify and detect; 2) determine virulence; 3) determine drug resistance and other resistance markers from collected organisms; or a combination thereof. In particular embodiments, the invention provides compositions and methods for identifying, quantitating, and detecting influenza virus-specific nucleic acid segments within a population of polynucleotides obtained from a biological sample. In illustrative examples, a method has been developed to quickly and accurately identify the presence of Influenza A H1N1 (Swine flu 2009) viral strains in such a sample.

BACKGROUND

Numerous pathogens (e.g., viruses, bacteria, fungi, and parasites) cause infection and other illness in animal and human populations worldwide. Sometimes one or more mutations in a pathogen can cause a typical illness-causing pathogen to become a full-blown pandemic. Although these pathogens and resultant illnesses are varied, one of the more prominent based on current events is the influenza virus.

The influenza virus and its variations (collectively referred to herein as “the flu virus”) are the cause for a contagious respiratory illness (commonly referred to as “influenza,” “illness,” or the “Flu”) in humans and animals (interchangeably referred to herein as a “host,” “patient,” or “subject”) that can cause mild to severe illness, and at times can lead to death. Every year in the United States alone, on average: 5% to 20% of the population gets the Flu; more than 200,000 people are hospitalized from Flu complications—and about 36,000 people die from Flu.

The flu virus spreads in respiratory droplets typically transmitted through coughing and sneezing. In human patients, the virus usually spreads from person to person, though sometimes subjects become infected by touching something with flu viruses on it and then touching their mouth or nose. Most healthy adults may be able to infect others beginning 1 day before symptoms develop and up to 5 days after becoming sick. Uncomplicated influenza illness is often characterized by an abrupt onset of constitutional and respiratory signs and symptoms, including fever, myalgia, headache, malaise, nonproductive cough, sore throat, and rhinitis.

There are three main types of influenza viruses: influenza A, influenza B, and influenza C. Within each type of influenza and within influenza A in particular there are many different subtypes. These subtypes differ based upon certain proteins expressed on the surface of the virus, specifically the hemagglutinin (HA) and the neuraminidase (NA) proteins. To date sixteen HA subtypes and nine NA subtypes of influenza A virus have been reported from avian isolates. Many different combinations of HA and NA proteins are possible, however, and each combination represents a unique subtype.

“Human influenza virus” usually refers to those subtypes that spread widely among humans. There are three known influenza A subtypes currently circulating among humans: H1N1, H1N2, and H3N2, each of which include various individual influenza viral strains. Subtype H2N2, for example, (which includes strains often referred as ‘Asian Flu’ strains), circulated within the human population from 1957-1968. Subtype H1N1, which includes strains commonly referred to as ‘Swine Flu’ strains, is currently circulating within various human populations worldwide.

Alarmingly, because influenza A viruses can constantly undergo mutations, reassortments, genetic “drift and shift,” and the like, host specificities are changing. Influenza viruses can be spread among various animal species, and infection from non-human species (such as avians, porcines, primates, and other animals) to human hosts leads to new influenza subtypes that can adapt over time to infect and spread more rapidly or thoroughly among human populations. Examples that have been widely reported in recent years include, for example, H5, H7, and H9 subtypes.

The H5N1 subtype, for example, has been reported as having mutated sufficiently to spread from avian hosts to humans. While the spread of H5N1 virus from human to human has, at least for now, been limited, unfortunately that has not been the case with the spread of particular strains of the H1N1 subtype, which appear to be highly contagious, and infectious when spread from person to person. Additionally, because H5N1, H1N1, and many other subtypes have been historically less prevalent in human populations, there is little or no immune protection against them in humans at present. Indeed, it has been widely reported in the mainstream media, and commonly considered in the scientific community that, if virulent strains of Influenza A virus were to gain the capacity to spread easily from person to person, a worldwide outbreak of disease (i.e., pandemic) would likely ensue. The mid-summer 2009 declaration by the World Health Organization (WHO) that the spread of H1N1 influenza had reached Phase 6 pandemic proportion (nearly 100,000 laboratory-confirmed cases and over 400 deaths) in more than 120 countries, confirms this conventional wisdom.

Pandemic viruses typically emerge as a result of a process called “antigenic shift,” which causes an abrupt or sudden, major change in a virus, e.g., influenza A virus. In the process of antigenic shift, two or more different strains of a single virus (or of different viruses), combine to form a distinctly new subtype that expresses a unique combination of surface antigens found in the strains that originally combined. While antigenic shift has been reported in various viral species, it is most widely observed in influenza virus, thus representing the most common form of genetic reassortments that gives rise to a phenotypic change in the resultant strains.

With influenza, these changes are caused by influenza A viruses spread from birds and animals to humans, thereby creating new combinations of the HA and/or NA proteins on the surface of the virus. Such changes result in a new influenza A virus subtype. The appearance of a new influenza A virus subtype is the first step toward a pandemic. To cause a pandemic, however, the new virus subtype also would need the capacity to spread easily from person to person and be a subtype that is sufficiently dissimilar from the two typical strains (A and B) found in the human population. Once a new pandemic influenza virus emerges and spreads, it eventually becomes established and transmissible among human populations, circulating for many years as part of the seasonal epidemics of influenza.

While the extent and severity of a pandemic cannot be accurately predicted, several computer modeling studies suggest that the impact of a pandemic on the United States (and the world as a whole) could be substantial. In the absence of any control measures (e.g., vaccination or drugs), it has been estimated that a “medium-level” pandemic in the U.S. could cause 89,000 to 207,000 deaths, 314,000 to 734,000 hospitalizations, 18 to 42 million outpatient visits, and another 20 to 47 million incidents of illness. According to the Centers for Disease Control and Prevention (CDC), between 15% and 35% of the U.S. population could be affected by an influenza pandemic, with an economic impact estimated between approximately $70 and $170 billion. By summer 2009, CDC had reported more than 43,000 confined and probable cases of H1N1 in the U.S., with more than 300 of those resulting in death.

Biological organisms (also interchangeably referred to herein as “organisms” or “microorganisms”), such as bacteria and viruses, like influenza A, B, or a combination of organisms, and particularly pandemic influenza, threaten to quickly spread over large geographic ranges and through large populations, causing high rates of mortality and morbidity. Prior to mobilizing and implementing prevention tactics to ensure public health, it is critical to first and foremost detect and identify these organisms as soon as they appear. Early detection and surveillance to track the spread of such organisms might help mitigate the extensive damage predicted by the CDC in the event of a pandemic outbreak, e.g., influenza. Early detection is also expected to be critical in limiting or helping to treat the damage from any biological terrorism. Thus, a system to rapidly detect and identify organisms is most desirable.

Conventional techniques to detect and identify viruses, however, are not suitable for this task. Generally virus surveillance, detection and identification are time consuming (e.g., days to weeks, and in some cases, months), cumbersome to conduct, and have the potential of posing numerous health risks to health care personnel and even the general public. Most techniques typically require cold chain cultures (with safety level 3 to 4 protocols), which is associated with fairly high levels of risk. The conventional surveillance, detection, and identification process (collectively referred to herein as “the surveillance process”) typically includes culturing a live target specimen (interchangeably referred to herein as “targeted specimen,” “tissue,” or “sample”), such as bird, swine, human, or other living cells; transporting the sample to a suitable laboratory facility or other testing site, such as national, regional, or state testing laboratories; and then testing the target specimen for a range of biological organisms. Based on assays of genomic material (e.g. RNA and/or DNA) in the target sample, the organism(s) can often be identified.

Inherent in this identification and detection process is the need for bringing the target specimen back to a laboratory, thereby adding time and risk to the entire process. If the target specimen is found remotely, then it must be carefully transported to a suitable diagnostic laboratory so as to not harm, contaminate, or risk accidental exposure of the specimen—of the people handling the specimen during transport. During transportation, for example, the specimen is typically kept in a refrigerated or near frozen condition to ensure that the specimen is kept alive and the tissues to be tested remain intact.

Thus, Applicants have discovered a need in the art for a simple to use, stable, rapid diagnostic tool and product that, rather than culturing an organism and/or sending the specimen to a remote laboratory, would allow more rapid detection and identification of biological organisms, such as microorganisms (e.g., viruses and bacteria), at or adjacent a specimen collection site. The diagnostic tool should be portable and capable of being operated remotely from a conventional laboratory, and preferably would provide safety in such an environment compared to conventional diagnostic methods used in regional facilities, such as culturing such organisms.

SUMMARY OF THE INVENTION

The present invention meets unmet needs in the art by providing an inventive diagnostic product (also interchangeably referred to herein as a “biological organism identification product” and a “diagnostic tool”), and methods of using the same, to rapidly detect and identify microorganisms. In particular applications the diagnostic product permits the collection of a target specimen, preparation of the target specimen for assaying, isolation of genomic material, and subsequent processing of the genomic material to identify the organism. Generally, the diagnostic tool can be used in the field to collect one or more organisms and identify the collected organism(s), and provides a relatively immediate form of surveillance against potential epidemics, outbreaks, infections, and other biological organisms of interest.

Embodiments of the present invention encompass a biological organism identification product that includes a collection device to collect one or more sample organisms, a fixing and transporting composition present in an amount sufficient to kill one or more sample organisms associated with the collection device, an extraction member to extract a sufficient amount of genomic nucleic acid from one or more sample organisms to facilitate identification thereof, and a stabilized polymerase chain reaction (PCR) component into which the sufficient amount of genomic nucleic acid can be dissolved.

Preferred embodiments of the present invention include a durable, stand-alone biological organism product (referred to interchangeably herein as a “kit”) that can conduct a plurality of field diagnoses. The kit may preferably include a portable enclosure to retain the product components including the collection device, fixing and transporting composition, extraction member, and a stabilized component. The kit may also include machinery to conduct the PCR and/or a power source or power adapted to operate any machinery. In certain embodiments, the diagnostic kit also includes a plurality of active pharmaceutical ingredient doses in an amount sufficient to prevent or treat one or more conditions caused by the identified biological organism.

The present invention, in certain embodiments, relates to methods of identifying a biological organism that includes collecting a biological sample from a subject, fixing the biological sample in a sufficient amount of a fixing agent to minimize or eliminate any contamination by the biological sample, extracting a sufficient amount of genomic nucleic acid from the fixed biological sample, and assaying the sufficient amount of the genomic nucleic acid in a lyophilized polymerase chain reaction component to obtain information about the organism. In preferred embodiments, the polymerase chain reaction component has a sufficient amount of one or more primers, which identify predetermined organisms and each of which is chemically associable to a protein component specific to a biological organism. Preferably, this all occurs in a single location.

In other embodiments, the method is relatively rapid compared to conventional organism detection techniques. In some embodiments of the method, no more than about 24 to 72 hours pass from the collecting of the target specimen to the assaying of the genomic material to obtain identification information. In some embodiments of the invention, the assaying is conducted for about 30 to 180 minutes, preferably 45 minutes to 150 minutes.

The invention also encompasses a reagent mix for detection of a microbial sequence, the reagent mix including one or more microbe-specific primers, probes, or enzymes, or a combination thereof, present in a mixture that is at least substantially stable at room temperature and is adapted and configured for use with a polymerase chain reaction (PCR) device. In one embodiment, the reagent mix is substantially stable at room temperature for at least about 5 days and up to two weeks. In another embodiment, the detection of the microbial sequence occurs within about 90 minutes after the microbial sequence is extracted from a sample. The reagent mix can be used to identify a microbial sequence, such as a pathogen, bacterial or viral sequence, or combination thereof. The reagent mix of the present invention, also referred to herein as a “prime mix,” can also be used to identify strains of a viral or bacterial sequence, or even sub-strains of influenza.

In another embodiment, the reagent mix can be used as part of an apparatus to facilitate determination of a microbial amino acid sequence. In preferred embodiments of the invention, the reagent mix is particularly suited to field use, and can be used in conjunction with a collection device that collects one or more biological organism samples. In additional embodiments, identification of the same occurs within about 90 minutes.

A further embodiment of the invention includes a method for detection of a microbial sequence that includes obtaining genomic nucleic acid from a biological sample and assaying the genomic material by adding the nucleic acid to the reagent mix of one or more microbe-specific primers, probes, or enzymes, or a combination thereof, wherein the mix is substantially stable at room temperature and is adapted for use with a PCR device. In another embodiment, the PCR device includes fluorescence detection equipment for real-time PCR detection.

In one embodiment, the invention provides a method for detecting the presence or absence of an Influenza virus-specific nucleic acid segment, and in particular aspects, provides a method for detecting the presence or absence of a particular type, subtype, or strain of Influenza virus. In exemplary embodiments, the invention provides a method of identifying an Influenza A H1N1 subtype virus-specific nucleic acid segment in a population of polynucleotides that is preferably obtained from a biological sample.

In an overall and general sense the method includes performing at least one cycling step, wherein the cycling step includes at least a first amplifying and at least a first hybridizing, wherein the at least a first amplifying includes contacting the sample with a pair of Influenza A H1N1 subtype virus-specific amplification primers to produce an Influenza A H1N1 subtype virus-specific amplification product if an Influenza A H1N1 subtype virus-specific nucleic acid segment is present in the sample; and wherein the at least a first hybridizing is accomplished using a labeled detection probe that is specific for the amplification product, wherein the presence of the amplification product is indicative of the presence of one or more Influenza A H1N1-specific nucleic acid segments in the population of polynucleotides.

In particular aspects, the pair of amplification primers includes a first oligonucleotide primer of less than about 50, preferably less than about 40, and more preferably still, less than about 30 nucleotides in length that comprises, consists essentially of, or alternatively, consists of the nucleic acid sequence 5′-AGCCTYCCATTTCAGAATATACA-3′ (SEQ ID NO:51).

Likewise, in certain aspects, the pair of amplification primers includes a second oligonucleotide primer of less than about 50, preferably less than about 40, and more preferably still, less than about 30 nucleotides in length that comprises, consists essentially of, or alternatively, consists of, the nucleic acid sequence 5′-AATCCTGTRGCCAGTCTCAATTTTG-3′ (SEQ ID NO:52).

In certain illustrative aspects, the presence of an amplification product so produced in an amplification of the subject population of polynucleotides (such as for example, by using PCR-based amplification methodologies) may be detected through the use of a labeled oligonucleotide probe that is specific for the amplification product so produced. In illustrative examples presented herein, the detection probe includes a first oligonucleotide probe of less than about 50 nucleotides in length, preferably of less than about 40 nucleotides in length, and more preferably still of less than about 30 nucleotides in length, and further wherein the detection probe includes a nucleic acid sequence that comprises, consists essentially of, or alternatively consists of, the nucleic acid sequence of 5′-TCCAAAATATGTAAAAAG-3′ (SEQ ID NO:53).

In a related embodiment, the method may be employed using a composition that includes (a) a pair of amplification primers includes: (i) a first oligonucleotide primer of less than about 50 nucleotides in length that includes the nucleic acid sequence of SEQ ID NO:51, and (ii) a second oligonucleotide primer of less than about 50 nucleotides in length that includes the nucleic acid sequence of SEQ ID NO:52; and (b) a first detection probe of less than about 50 nucleotides in length that specifically binds to the amplification product so produced, and that includes a nucleic acid sequence that comprises, consists essentially of, or alternatively consists of the sequence of SEQ ID NO:53.

In the practice of the invention, the population of polynucleotides so analyzed will preferably be obtained from a biological sample, with biological samples obtained from a mammal (including e.g., humans, non-human primates, domesticated livestock, and the like). Exemplary biological samples include, without limitation, one or more samples selected from the group consisting of blood, plasma, cells, tissues, and serum. Samples may be obtained at any time prior to the amplification protocol, and subsequent detection of amplification products, but in particular aspects, the time between sample collection, isolation of a population of polynucleotides from the sample, and the amplification/detection analysis of the target nucleic acids of interest is quite short, such as, on the order of minutes to hours from specimen collection to amplification product detection. In certain embodiments, the composition may further include one or more specimen stabilizing, inactivating, storage, transport solutions, as well as one or more PCR buffers, reagents, polymerases, and such like.

In particular aspects as described herein, in certain situations, particularly when performing the method for the analysis of specimens that are acquired in remote or field sites, the compositions of the present invention are preferably stable for extended periods of time at ambient temperatures, and the collected biological specimens do not require particular refrigeration or freezing in order to prepare them for the amplification/detection steps of the overall identification process.

It is contemplated that in certain embodiments, the compositions disclosed herein may be formulated such that the entire specimen collection and nucleic acid amplification/detection process may be accomplished in remote, field, battlefield, rural, or otherwise non-laboratory conditions without significantly limiting the fidelity, accuracy, or efficiency of the amplification/detection methodology. Such aspects of the invention provide particular advantages over conventional laborious isolation/collection/transport/storage/analysis protocols that require several days to several weeks to achieve, and must often be conducted under conditions that require refrigeration or freezing of the sample and/or assay reagents in order to properly complete the analysis. By providing reagent mixtures that include all of the necessary isolation, storage, and polynucleotide stabilization components, as well as all of the necessary reagents for amplification of selected target nucleotides (including, without limitation, the amplification primers and detection probes described herein, alone or in combination with one or more PCR buffers, diluents, reagents, polymerases, detectable labels, and such like) in a single, shelf-stable, ambient-temperature facile reagent mix, significant cost savings, time-reduction, and other economies of scale may be achieved using the present invention as compared to many of the conventional oligonucleotide probe-based thermal cycling assays currently available in the marketplace. The detailed use of particular isolation/storage/transport solutions that are contemplated to be applicable to the preparation of target populations of polynucleotides is described in copending U.S. patent application Ser. No. 12/243,949, filed Oct. 1, 2008, which is commonly co-owned with the present application, and the contents of which is specifically incorporated herein in its entirety by express reference thereto.

When a real-time PCR methodology is employed for the amplification, the detecting may optionally performed at the end of a given number of cycles, or alternatively, after one or more of each cycling step in the amplification protocol.

In the regular practice of the method, one may also perform the cycling step on one or more “negative” and/or “positive” control sample(s) as is routinely done in the molecular genetic assay arts to ensure integrity, fidelity, and accuracy of the method. The use of such controls is routine to those of ordinary skill in the art and need not be further described herein. Likewise, in the practice of the invention, it may also be desirable to incorporate one or more known “internal positive controls” into the population of polynucleotides to be isolated, to further ensure the integrity, fidelity, and/or accuracy of the disclosed method. The detailed use of such controls is described in copending U.S. patent application Ser. No. 12/426,890, filed Apr. 20, 2009, which is commonly co-owned with the present application, and the contents of which is specifically incorporated herein in its entirety by express reference thereto.

In another embodiments, the invention provides an Influenza A H1N1 Virus-specific oligonucleotide amplification primer set, wherein the first amplification primer is less than about 50 nucleotides in length and includes the nucleotide sequence of SEQ ID NO:51 and the second amplification primer is less than about 50 nucleotides in length and includes the nucleotide sequence of SEQ ID NO:52. This Influenza A H1N1 Virus-specific oligonucleotide amplification set may optionally further include a first detection probe, wherein the first detection probe includes a labeled oligonucleotide probe of less than about 50 nucleotides in length that includes the nucleotide sequence of SEQ ID NO:53.

The invention also provides a diagnostic nucleic acid amplification/detection kit that generally includes, in a suitable container, an Influenza A H1N1 Virus-specific oligonucleotide amplification primer set as described herein, and instructions for using the primer set in a PCR amplification of a population of polynucleotides obtained from a biological sample or specimen. Such kits may further optionally include, in the same, or in distinct containers, an oligonucleotide detection probe that specifically binds to the amplification product produced from PCR amplification of a population of polynucleotides obtained from a biological sample or specimen that contains, or is suspected of containing, an Influenza A H1N1 Virus-specific nucleic acid segment. Such kits may also further optionally include, in the same, or in a distinct container, any one or more of the reagents, diluents, enzymes, detectable labels (including without limitation, one or more radioactive, luminescent, chemiluminescent, fluorescent, enzymatic, magnetic, or spin-resonance labels), dNTPs, and such like that may be required to perform one or more thermal cycling amplifications of a population of polynucleotides as described herein. The kits, may also further optionally include, in the same, or in a distinct container, any one or more buffers, surfactants, chaotropes, DNAses, RNAses, or other such nucleic acid isolation and/or purification reagents as may be required to prepare a sample for analysis. In certain embodiments, the kits of the invention may also optionally further include one or more portable, ruggedized, or field-employable thermal cycling, PCR amplification systems and/or one or more systems, devices, or instruments to facilitate detection, quantitation, and/or distribution of the detectable label(s) employed for visualization of the amplification products produced during the practice of the method.

In another embodiment, the invention provides an article of manufacture that includes a pair of Influenza A H1N1 Virus-specific oligonucleotide amplification primers; and a first Influenza A H1N1 Virus-specific oligonucleotide detection probe; wherein the detection probe includes at least one detectable label. Such article of manufacture may optionally further include, for example, one or more package insert(s) having instructions for using the pair of primers and the detection probe to detect the presence or absence of an Influenza A H1N1 Virus-specific nucleic acid segment within a population of polynucleotides obtained from a biological sample that was collected from a human subject.

In yet another aspect, the invention also provides a composition that includes:

(a) a first pair of Influenza A H1N1 Virus-specific amplification primers, wherein the pair of primers includes:

(i) a first oligonucleotide primer of less than about 50 nucleotides in length that includes the nucleic acid sequence of SEQ ID NO:51; and

(ii) a second oligonucleotide primer of less than about 50 nucleotides in length that includes the nucleic acid sequence of SEQ ID NO:52; and

(b) a first Influenza A H1N1 Virus-specific oligonucleotide detection probe, including:

(i) a first oligonucleotide detection probe of less than about 50 nucleotides in length, wherein the probe includes the nucleic acid sequence of SEQ ID NO:53; and

(ii) at least a first detection reagent operably linked to the oligonucleotide detection probe.

In yet another embodiment, the invention provides a method of identifying a subtype of Influenza A virus. This method generally involves detecting the presence of an Influenza A virus-specific nucleic acid segment in a population of polynucleotides, if present, using a labeled oligonucleotide probe that is specific for the Influenza A virus-specific nucleic acid segment; and if an Influenza A virus-specific nucleic acid segment is present in the population, then further identifying the Influenza virus using a labeled oligonucleotide probe that is specific for an H5 or an H1N1 subtype of the virus. Such method may further optionally include the additional step of determining whether the influenza A type-specific nucleic acid segment so detected is specific for a particular subtype, or specific for a particular strain of influenza virus, such as, for example, whether the influenza A type-specific nucleic acid is an H5 or an H1N1 subtype (such as an H1N1 Swine 2009) subtype.

In such methods, the identification and/or characterization of the particular subtype of Influenza A may be performed simultaneously, or sequentially. In other embodiments, it may also be desirable to determine the presence or absence of Influenza B-specific nucleic acid sequences in the population of polynucleotides so analyzed. In such cases, an assay that is specific for Influenza B may also be performed on the sample either simultaneously or sequentially.

In illustrative examples, the labeled oligonucleotide probe useful in detecting particular Influenza sub-type or strain-specific nucleic acid sequences will preferably comprise, consist essentially of, or alternatively consist of, a labeled oligonucleotide probe that comprises the sequence: 5′-TCCAAAATATGTAAAAAG-3′ (SEQ ID NO:53), 5′-TCAGGCCCCCTCAAAGC-3′ (SEQ ID NO:54); 5′-ATGGGAAATTCAGCTCT-3′(SEQ ID NO:55); 5′-TCTCCAAAGTATGTCAGG-3′ (SEQ ID NO:56); 5′-TGAGATCAGATGCACCCAT-3′ (SEQ ID NO:57); 5′-AGAGRGGAAATAAGTGG-3′ (SEQ ID NO:58), or any combination thereof. As described elsewhere herein, such oligonucleotide probes are preferably labeled with a fluorescently-detectable label, including, without limitation, FAM.

In another embodiment, the invention provides a composition for the detection of nucleic acids that is at least substantially stable at room temperature and is adapted and configured for use with a polymerase chain reaction (PCR) device.

The invention also provides a biological organism identification product that generally includes: (a) a collection device to collect one or more sample organisms; (b) a fixing and transporting composition present in an amount sufficient to kill the one or more sample organisms associated with the collection device; (c) an extraction member to extract a sufficient amount of genomic nucleic acid from the one or more sample organisms to facilitate identification thereof; and (d) a substantially stable polymerase chain reaction (PCR) component into which the sufficient amount of genomic nucleic acid can be added. In exemplary embodiments, the PCR component will preferably include a composition that includes (a) a first pair of Influenza A H1N1 Virus-specific amplification primers, wherein the pair of primers includes: (i) a first oligonucleotide primer of less than about 50 nucleotides in length that includes the nucleic acid sequence of SEQ ID NO:51; and (ii) a second oligonucleotide primer of less than about 50 nucleotides in length that includes the nucleic acid sequence of SEQ ID NO:52; and a first Influenza A H1N1 Virus-specific oligonucleotide detection probe, that includes: (i) a first oligonucleotide detection probe of less than about 50 nucleotides in length, wherein the probe includes the nucleic acid sequence of SEQ ID NO:53; and (ii) at least a first detection reagent operably linked to the oligonucleotide detection probe.

In another embodiment, the reagent mix is configured and adapted used to identify one or more sample biological organisms that have been collected by a collection device. In a preferred embodiment, the reagent mix is used to identify the one or more samples at a field site or a remote location.

In another embodiment, the reagent mix is contained in a liquid form. In a preferred embodiment, the reagent mix is present in a liquid form in a test tube, a 96-well plate, or a capillary vessel. In yet another embodiment, the mixture is lyophilized.

The invention further encompasses methods for detecting a microbial sequence which includes: obtaining genomic nucleic acid from a biological sample, and assaying the genomic nucleic acid by adding the nucleic acid to the reagent mix, wherein the mix is at least substantially stable at room temperature and is configured and adapted for use with a polymerase chain reaction (PCR) device. In another embodiment, the assaying further includes adding the reagent mix to the polymerase chain reaction (PCR) device, running the assay, and completing the assay in less than about 90 minutes. In a preferred embodiment, the assaying further includes detecting the microbial sequence in real-time using fluorescence equipment adapted for use with the PCR device. In yet another embodiment, the genomic material is from a bacteria or virus, or a pathogen. In yet a further embodiment, the genomic material is from an influenza virus.

Commercial Formulations and Kits

The present invention also provides kits and sample collection systems utilizing the disclosed compositions described herein. In particular embodiments, such sample collection systems may include a collection device, such as a swab, curette, or culture loop; and a collection vessel, such as a vial test tube, or specimen cup, that contains one or more of the compositions disclosed herein. The collection vessel is preferably releasably openable, such that it can be opened to insert the one-step compositions and closed and packaged, opened to insert the sample and optionally a portion of the collection device and closed for storage and transport, or both. The collection vessel may use any suitable releasably openable mechanism, including without limitation a screw cap, snap top, press-and-turn top, or the like. Such systems may also further optionally include one or more additional reagents, storage devices, transport devices, and/or instructions for obtaining, collecting, transporting, or assaying one or more samples in such systems.

Kits may also be packaged for commercial distribution, and may further optionally include one or more collection, delivery, transportation, or storage devices for sample or specimen collection, handling, or processing. The container(s) for such kits may typically include at least one vial, test tube, flask, bottle, specimen cup, or other container, into which the composition(s) may be placed, and, preferably, suitably aliquoted for individual specimen collection, transport, and storage. The kit may also include a larger container, such as a case, that includes the containers noted above, along with other equipment, instructions, and the like. The kit may also optionally include one or more additional reagents, buffers, or compounds, and may also further optionally include instructions for use of the kit in the collection of a clinical, diagnostic, environmental, or forensic sample, as well as instructions for the storage and transport of such a sample once placed in one or more of the disclosed compositions.

Any of the embodiments illustrated herein stand independently, and any features or embodiments may be combined in any way, unless expressly excluded, to achieve a preferred embodiment. Additional advantages and embodiments of the invention will also become more apparent to those of ordinary skill in the art upon review of the teachings of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

For promoting an understanding of the principles of the invention, reference will now be made to the embodiments, or examples, illustrated in the drawings and specific language will be used to describe the same. It will, nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one of ordinary skill in the art to which the invention relates.

The following drawings form part of the present specification and are included to demonstrate certain aspects of the present invention. The invention may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:

FIG. 1A and FIG. 1B illustrate the stability of reagents over time at varying temperatures in accordance with one embodiment of the invention;

FIG. 2A, FIG. 2B, and FIG. 2C illustrate the stability of the reagents over time at varying temperatures in accordance with one embodiment of the invention;

FIG. 3 illustrates the amplification and detection of an Influenza B-specific nucleic acid sequence according to an embodiment of the present invention;

FIG. 4A shows a nucleic acid sequence alignment from the H5 region of 22 Influenza A viral strains, and the particular forward and reverse primer sequences utilized for amplification of the H5 target consensus;

FIG. 4B shows C_T data obtained from an Influenza A H5-specific thermal cycling assay;

FIG. 5 shows Table 1, which presents exemplary oligonucleotide sequences for various primer/probe sequences used in the real-time RT-PCR amplification assays described herein, as well as the forward and reverse primer sequences used for the in vitro generation of cDNA target templates for H1, H3, and H5-specific isolates;

FIG. 6 summarizes the data from Table 2, which illustrates the detection of various influenza virus type A and B strains using type-specific assays;

FIG. 7 summarizes the data from Table 3, which illustrates the detection of various influenza virus types (A/B) and subtypes (H1, H3, and H5) in cultured clinical isolates using real-time RT-PCR;

FIG. 8 summarizes the data from Table 4, which illustrates the detection of various influenza virus types (A/B) and subtypes (H1, H3, and H5) in uncultured primary clinical specimens using real-time RT-PCR;

FIG. 9 summarizes the data from Table 5, which illustrates the primer/probe sequences used for real-time RT-PCR amplification and in vitro generation of cDNA target templates for the H1N1 (Swine flu 2009) subtype of Influenza A virus;

FIG. 10 depicts the real-time RT-PCR analysis of eight human clinical nasal wash samples preserved in PrimeMix™ Solution that includes primers and probe sequences for H1 swine 2009 strains (hereinafter “PrimeMix Swine H1”). Three of the eight samples tested positive for a H1 swine 2009 strain, as indicated by the real-time RT-PCR CT values.

FIG. 11 depicts the 100-fold titration of a H1N1 positive human clinical sample when tested using universal Influenza A-specific primers and probe as well as the Swine flu 2009 H1N1-specific primers and probe in RT-PCR reactions. Both assays detected Influenza A RNA with similar amplification curves and C_T values.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides a diagnostic tool, and methods of using the same, that permit rapid identification of one or more biological organisms of interest. Preferably, the detection and identification is sufficiently rapid so as to permit real-time or substantially real-time surveillance as to the spread of a particular organism in one or more host populations. In particular, the invention can combine recombinant DNA/RNA isolation and detection techniques to rapidly and remotely detect and identify organisms, such as microorganisms, typically pathogens. The pathogens most typically in need of identification according to the invention include microbes that cause malaria, viruses, preferably communicable viruses, and more preferably influenza, and bacteria. Advantageously, the identification can further include sub-typing and/or lineage distinction of an influenza strain or a similar species identification of another microorganism. Typically the sample is collected from a host, which provides the sample to be tested in the diagnostic product of the invention. More specifically, the present invention can advantageously allow isolation of the organism from host tissue, isolation of the genomic material of the organism, detection and identification of organisms from a target sample, on-site analysis of the organism, and identification of the organism. In some embodiments, the diagnostic tool includes a therapeutic, preventative, or prophylactic agent for administration to the host(s), which agent is selected based on the organism identified according to the invention. In other embodiments, the diagnostic tool includes detecting resistance to therapeutic agents.

The present invention provides advantages over the prior art by providing more rapid and efficient detection, classification/sub-typing, and isolation of biological organisms from a host or target specimen. In variations of the present invention, the components of the diagnostic tool can be securely enclosed in a portable enclosure to retain them in association for travel to a field site, or for use in emergency rooms and doctor's offices.

Preferred embodiments of the invention allow the identification of a biological organism at a field site. Advantageously, the enclosure contains sufficient equipment to permit multiple identifications of different collection samples in the field without requiring transport or return to a laboratory or central processing center. As used herein, the “field” encompasses any setting outside of the traditional laboratory setting. This includes emergency rooms and doctor's offices, as well as the outdoors, villages, homes, commercial offices, warehouses, streets, field hospitals, etc., and areas in which there are limited or no modern amenities (e.g., portable water and/or electricity).

In some embodiments of the present invention, the diagnostic tool includes equipment and materials to conduct and analyze the PCR for each assay. The machinery to conduct PCR is readily understood by those of ordinary skill in the art, and lower weight and bulk selections can be made according to the invention as desired to increase portability of the kit of the invention. The assays can be used in association with many types of PCR instruments, preferably with virtually every PCR instrument. Preferably, the PCR equipment is sufficiently light-weight, and adapted to draw minimal power, for increased portability and duration of use. Fluorescence-linked PCR equipment for real-time identification of a microbial sample, as is known in the art, can also be used. Although any suitable PCR equipment may be included in the product of the invention, one preferred type of PCR equipment includes the field-hardened R.A.P.I.D.® PCR equipment commercially available from Idaho Technology. Other commercially available instruments that can be used in accordance with the present invention include the LightCycler®, and the ABI 7500 (7000).

In yet other embodiments of the present invention, particularly where PCR equipment is included in association with the portable enclosure, the portable enclosure includes at least one power source. While any suitable power source providing sufficiently consistent electrical output may be used, preferably the power source includes a battery, an electrical generator, a solar panel, or a combination thereof, along with any associated devices such as power cords or plug adapters to facilitate connection of the power source to any field equipment, such as a PCR device, that requires electricity to conduct the methods of the invention. In some variations of the present invention, the diagnostic product further includes replacement or repair components to maintain or enhance operation of the diagnostic tool over extended periods of time in the field without resupply. This feature is essential in some embodiments, as the product of the invention may be used in a quarantine or restricted travel environment where fresh supplies may not be available. In additional embodiments, the diagnostic tool includes any desired processor, e.g., a computer or a PDA with the relevant software, to conduct an analysis of the diagnostic testing. In certain variations, the processor determines which, if any, organisms are identified and detected. The software can be adapted and configured to search for certain likely types of organisms first depending on the field location, e.g., malaria in a tropical setting, as well as providing a knowledge base of biological organisms with information that can permit an optional, associated human operator to make any desired adjustments or to assist with the detection and identification, e.g., an analysis of the assay results.

As used herein, the term “infection,” “influenza infection,” “viral infection,” “bacterial infection,” and the like are used consistently with their accepted meanings in the art, but can also encompass the detrimental effect of a biological organism that does not result in an infection as conventionally understood. The term “methods of treating” includes methods of managing, and when used in connection with the biological organism or infection, includes the amelioration, elimination, reduction, prevention, or other relief or management from the detrimental effects of a biological organism. In a preferred embodiment, these detrimental effects include an influenza infection, influenza virus, symptoms characterizing and/or effects associated with influenza in the subject, or a combination.

In some embodiments of the present invention, the portable diagnostic is equipped with suitable amounts of components to conduct a plurality of assays, without leaving the field or compromising sterilization or a quarantine. In some variations, the portable enclosure includes each selected component to detect and/or identify an organism in an amount sufficient to conduct at least 10 field diagnoses, preferably about 20 field diagnoses, more preferably about 50-field diagnoses, and most preferably about 100 field diagnoses. In preferred variations, the portable enclosure includes at least enough components to conduct a plurality of field analyses while maintaining the portability of the diagnostic tool. Another measure of the quantity of components present is enough of each of the types of components necessary to identify an organism over an extended period of time in the field. For example, this time period might be about 6 hours to 2 weeks, preferably 12 hours to 1 week.

In other preferred embodiments, the diagnostic tool is durable and stable, particularly during storage and transport, as well as preferably in the field during use. The components of the diagnostic tool are hardened to resist degradation even at storage temperatures of about 18° C. to 27° C., and preferably from about 20° C. to 25° C.

In addition to temperature resistance, the portable enclosure and the contents are preferably also adapted and configured to resist or prevent physical damage and/or breakage of the components therein according to the invention. The enclosure need not be complete, and may include gaps, holes, or projections to facilitate transport or storage. The product can also be arranged in modular form, such as in blister packs, so that each type of component can be stored separately or together if desired. For example, each module can hold all the components of the product, or each module can hold one type of component. Preferably, one module includes the stabilized component(s) and is stored separately under chilled conditions (i.e. less than room temperature), such as under refrigeration of less than 4° C., or more preferably under frozen conditions, until the product is ready to be transported to a remote field site. The chilled module can be combined or included with the other modular components by any conventional means to form the complete product.

Preferably, it is a complete enclosure sufficient to resist water penetration, and the enclosure is preferably waterproof. In some variations, the portable enclosure optionally includes an apparatus to facilitate portability, such as a handle, a strap, etc. In other variations, the portable enclosure includes a fastenable and resealable opening and closing feature that allows access to the components, safe storage of the portable enclosure, and/or maintenance of sterilized components, e.g., one or more door or hatch handles, zippers, latches, or the like.

The enclosure itself may be made of any sufficiently resilient packaging material available to those of ordinary skill in the art that can protect fragile contents such as glassware or PCR equipment or otherwise help increase the integrity of the components of the product, particularly any lyophilized reagents or samples that are present. Preferably, the enclosure and components are made of a suitable non-breakable material other than glass to facilitate shipment or transport of the enclosure to a field site. Examples of such packaging material that can be included in forming the portable enclosure include: aluminum or plastic foil, blister packs, cardboard or other paperboard, or a polymeric or other plastic component such as a thermoplastic polyolefin, or a temperature-stable polymer. For example, the resilient packaging material may include a temperature-stable polymer such as a propylene homopolymer or a copolymer of at least 50 mole percent of propylene and at least one other C₂ to C₂₀ alpha-olefin, or mixtures thereof. Exemplary alpha-olefins of such copolymers include ethylene, 1-butene, 1-pentene, 1-hexene, methyl-1-butenes, methyl-1-pentenes, 1-octene and 1-decene, or a combination thereof.

The enclosure may be formed through any available process, which may be selected by one of ordinary skill in the art with reference to the type of material. For example, a polymeric material may be molded or extruded. While any shape may be imparted to the enclosure that is sufficient to enclose the components, preferably the enclosure has a base that is sufficiently stable (e.g., flat) that it will not tip over during storage or use. The enclosure can be arranged to fold open to a table if desired, with the legs folded up inside when closed and an outer surface of the enclosure forming the tabletop surface when opened. The contents may be placed on the opened table to provide a convenient workbench when conducting the methods of the invention. Other convenient arrangements of the enclosure can be envisioned, such as an opening to form a shelf that can be placed on an existing table.

The diagnostic product includes one or more types of collection device to capture, collect, or otherwise take the target specimen from the host, and then contain or hold the specimen for further analysis. The specimen may include tissue, blood, saliva, or another biological product testable for genomic material from microorganisms present in the specimen. Any suitable collection device may be used to accomplish this goal. For example, swabs may be used to collect mucosal samples. Samples are preferably collected from the skin, nasal passages, oral passages, or a combination thereof. If necessary, blood may be drawn to obtain the necessary sample. Preferably, the collection device is sterile. Other preferred collection devices are those compatible for analysis with processing machinery, assay compositions and volumes, and/or size requirements for portability or as provided by the diagnostic kit described herein. Sufficient numbers of collection device, as further discussed herein, should be included to permit use of the diagnostic product in the field for an extended period of time in the event of a crisis.

In a preferred embodiment, the target specimen, or tissues or cells thereof, are immediately preserved upon, or shortly after, collection. Preferably, the target specimen is treated to kill the biological organism(s) contained therein. A preferred embodiment of the invention includes a fixing and transporting composition, which is typically a liquid and preferably a solution, emulsion, or suspension. The fixing and transporting composition helps minimize or eliminate contamination of the sample or the environment, as well as inhibiting or preventing escape of the sample. Preferably, the fixing and transporting composition includes alcohol (e.g., ethanol), guanidinium thiocyanate, or a combination thereof. Any suitable fixing and transporting composition (also referred to herein as a “fixing and transporting agent”), may be used to kill (i.e., fix) the organisms by disrupting a cellular membrane in the organism. The specimen may then be more safely transported to the assay site, which can be across a room from a patient, in a nearby room, or even more remote such as across the street or in a different part of the field site. For example, collection of genomic material from patients may occur in one tent or room, while the assays and PCR equipment are located nearby, such as within a few minutes drive. The collection device may be dried after being exposed to the fixing and transporting composition, but preferably the collector remains in the composition until just prior to the assay.

The collection and fixing of the target specimen may be arranged as follows. A cotton-tipped swab can be contacted with the nasal passages of a host. The organism(s) collected are then fixed. One fixing step that can be carried out in accordance with the present invention is generally described in Krafft, A. E., et al., Evaluation of PCR Testing of Ethanol-Fixed Nasal Swab Specimens as Augmented Surveillance Strategy for Influenza Virus and Adenovirus Identification, (J. Clin. Microbiol, 43(4):1768-1775, April 2005) which is specifically incorporated herein in its entirety by express reference thereto. Another method of accomplishing the fixing is reported in Chomczynski and Sacchi (Anal. Biochem., 162:156-159, 1987), through the use guanidinium thiocyanate.

The swab is either soaked in or placed in alcohol to kill the organism cells while sufficiently preserving the specimen for analysis. As used herein, “preserve” means that the nucleic acid material of the organism is not unduly damaged in the fixing process so that an assay and identification can be conducted. The fixing results in a significantly safer specimen that does not require cold-temperatures for preservation, such that the non-living specimen can be transported and even shipped, via standard postal mail if necessary. Further, because the specimen is killed, there is no risk of further outbreak or infection in the carrier or those associated with shipment of the sample if necessary. For example, even though the entire method can be performed in the field, it may be desired to conduct the assay and identification in a laboratory, either in the first instance or as a second trial to confirm the results of the field identification. In a preferred embodiment, the fixing and transport composition encompasses a non-hazardous, fixed specimen that can be processed using the components of the diagnostic tool.

Moreover, a second collection device can be used and stored differently from the fixing and transporting composition. For example, a second swab could also be included in the collection device and used to collect a sample from a host for a second assay or a different type of assay, such as in a regional laboratory or on different equipment, to help confirm the diagnosis later. For example, one swab can be used to collect genomic material and assay the organism at the field site, while a second swab can collect genomic material and be disposed in a chilled package, such as a refrigeration or freezer unit for up to about 4 days, preferably capable of being transported to a remote laboratory to further analysis. The second swab can be used to help identify organisms and to test for new vaccine candidates. One example of portable, cold storage suitable for use with the invention is the American Thermal Wizard, available through American Thermal Wizard International.

The extraction member is used to extract genomic material, or other relevant biological material, to characterize and identify one or more organisms from the target specimen. As used herein, the “genomic material” includes nucleic acids, such as RNA and/or DNA, that provide information as would be known to one of ordinary skill in the art to facilitate identifying and characterizing an organism of interest. Buffers, centrifuges, syringes, etc., as would be known to one skilled in the art, are exemplary extraction members suitable for the present invention. Suitable extraction techniques include those generally described in Matthews, C. K., et al., Biochemistry, Second Edition, The Benjamin Cummings Publishing Co., 1996 and Tortora, G. J., et al., Microbiology: An Introduction, The Benjamin Cummings Publishing Co., 1992, which are incorporated herein by express reference thereto. Generally, the extracted genomic nucleic acid is present in an amount from about 0.1 microliters to about 10,000 microliters, more preferably from about 1 microliter to about 1000 microliters, and more preferably from about 10 microliters to 100 microliters. An exemplary amount of nucleic acid is 25 microliters.

With respect to the extraction member, and other devices and/or apparatus in the diagnostic tool, it is preferable to maintain the equipment and identification environment in sterile or uncontaminated form. The diagnostic tool may optionally, but preferably, include one or more components to sterilize or maintain sterilization as would be known to one skilled in the art in certain embodiments. The fixing agent may also be selected to provide suitable sterilization, which may be a desirable way to reduce the number of different optional components necessary to function effectively in the field.

In the present invention, the PCR component when preferably included in the product is preferably suitable for portability and field use and analysis. One exemplary PCR assay includes real time reverse transcriptase —PCR (rRT-PCR), as generally described in Das, A., et al., Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse transcriptase-PCR with Lyophilized Reagents, J. Clin. Microbiol., 44(9):3065-3073, September 2006; specifically incorporated herein in its entirety by express reference thereto.

In preferred embodiments, the pre-selected PCR reagents are premixed to include target primers and probes in one or more PCR-adapted vessels. In the most preferred embodiments, the vessels are adapted and configured to be compatible, operable and functional with the selected PCR machinery (i.e., the PCR device). For example, capillary pipettes that are sized and dimensioned to be operatively associated with the included PCR device may be directly inserted in the compatible PCR equipment for rapid use. In accordance with certain embodiments of the invention, these contain stabilized wet reagents adapted and configured for use with the genomic material in a PCR device. In one preferred embodiment, all of the PCR reagents are stabilized. The stabilized reagents can be already disposed in a PCR holding device to which a liquid including the extracted genomic material is later added. Alternatively, PCR-usable vessels may include stabilized materials or spherules that can universally fit various PCR machinery or stabilized PCR materials from the diagnostic product can be put into solution or other liquid containing the extracted genomic material. The solution is then added to the PCR holding device, which can then be placed in the PCR machinery. In one example, the PCR holding device contains the stabilized materials and the extracted genomic material in solution is added to it. Or, by way of another example, stabilized material can be added to a cuvette to which is added extracted genomic material in solution, or vice versa, and then the proper amount of that solution can be added into the PCR holding device (e.g., a pipette), and placed in the PCR machinery.

In another preferred embodiment of the invention, the desired PCR components used to contain and assay selected types of samples are pre-loaded into one or more vessels and are then stabilized to maintain the quality of the vessel and its contents for field use once the extracted genomic material is combined.

The PCR assay is preferably included in the product for field use, and encompasses detecting the genomic material of the organism. Accordingly, in preferred embodiments, at least one reagent of the PCR component includes one or more primers and/or one or more probes specific to the detection of one or more predetermined biological organisms. The diagnostic tool preferably includes primers predetermined and preselected for use in identifying certain specific organisms. As used herein, the primer is the composition used to detect specific genomic material, such as forward and reverse primers, and a probe is a sequence that binds to a microbial sequence for amplification. The PCR provides amplified genomic material, which can chemically associate with certain primers and/or probes. In some embodiments, the primer, probe, or combination can include an anti-sense nucleic acid sequence that is chemically associable with the genetic material of a detected organism. In other embodiments, the primer, probe, or combination is chemically associable to a protein or component specific to the extracted genomic material from the biological organism. This specificity facilitates rapid identification of the organism, and/or sub-typing, e.g., to include influenza A subtypes H1, H3, H5, H7, H9, as may be readily determined by those of ordinary skill in the art particularly with reference to the present disclosure. For example, if a primer and/or probe specific to H5N1 influenza chemically associates in the assay, then there is evidence of the detection and identification of, e.g. H5 influenza in the targeted specimen.

The types of primers, probes, or both included in the diagnostic tool are preferably pre-selected by one of ordinary skill in the art. In some variations, a wide variety of primers, probes, or a combination thereof are included to detect and identify any of a corresponding wide variety of organisms, particularly where there is no advance knowledge of the type of organisms expected when using the diagnostic product. In situations in which a particular organism is suspected (e.g., H1N1 influenza), the range of primers, probes, or a combination thereof that are loaded into the portable enclosure may be focused on H1N1 influenza and influenza with a similar genomic composition, or may be exclusively those used for influenza. Preferably, the primers and probes specifically tailored to a particular strain do not cross react with other strains of the same or similar organism.

The PCR components may include a redundancy of primers and/or probes to ensure detection of a suspected organism and genome thereof. The library of primers and probes is generally increasing as the genomic sequence of new organisms are mapped, which can permit more suitable primer and probe selection for future uses of the diagnostic product. Descriptions of certain maps, primers, and probes that may be useful in connection with the invention includes those described in the Das publication, which is incorporated herein by express reference thereto. Preferably, the influenza primers and probes are designed to detect at least one strain of influenza encompassing the A or B types, and more preferably each of the sixteen H subtypes and each of the two B lineages.

In a particularly preferred embodiment, the PCR component is a reagent that includes one or more of amplification primers, detection probes and enzymes, or any combination thereof, present in a mixture. This mixture may further include standard PCR components such as water, buffer, nucleotides, polymerase, or the like, or any combination thereof. The mixture of standard components is known in the art as a master mix. One or more microbe-specific primers, probes, enzymes, or any combination can be added to the master mix to create the prime mix. The prime mix can have one, two, three or four or more microbial primers, probes and/or enzymes. The microbial primers, probes and/or enzymes can be specific to infective viral or bacterial agents in general, or to specific agents, such as those associated with influenza, dengue fever, malaria, HIV, SARS, MRSA, and tuberculosis. In one preferred embodiment, the primers, probes and/or enzymes in the prime mix are specific to the influenza strains A, B, or both. In another embodiment, the primers, probes, and/or enzymes are specific to sub-strains of influenza, such as H1, H3, H5, H7, and H9. In a further embodiment, the primers, probes, and/or enzymes are inclusive for all substrains of influenza (H1-H16 and N1-N9) and the two primary flu B circulating strains.

By way of example, certain primers and probes specific to influenza strains or types, or sub-strains or sub-types, are presented in FIG. 5 as well-suited to the present invention. The probes of FIG. 5 are oligonucleotide sequences located internal to the forward and reverse amplification primers. These oligonucleotides are dual labeled, containing one of several types of 5′ fluorescent reporters, e.g., 6-Carboxyfluorescein N-succinimidyl ester (FAM) and one of several types of 3′ quenchers, e.g., TAMRA, MGB Dark Quencher, etc. The sequences for influenza strains A and B are located on RNA Segment 7, which includes the open reading frames of the two matrix genes, M1 and M2, that are highly conserved among influenza virus strains. The sequences for influenza sub-types are located on RNA Segment 4, which codes from the hemagglutinin (HA) protein. Nucleotides “Y” and “R” are degenerative nucleotides that have been included in sequence positions that exhibit high variability, and represent mixtures of nucleotides “C and T” and “A and G”, respectively. Degenerative bases are used when there is genetic variability among strains at a particular nucleotide position within the genome.

In a further example, primers and probe sequences specific to the H1N1 influenza strain are presented in FIG. 9. These sequences were developed from a previously described real-time RT-PCR assay for the detection of contemporary circulating human H1 strains (Daum, et al., 2007). The probe of FIG. 9 is an oligonucleotide sequence located internal to the forward and reverse amplification primers. The probe is dual labeled, and contains a 5′ FAM fluorescent reporter and a 3′ Molecular-Groove Binding Non-fluorescence Quencher (MGBNFQ), which allows for the use of probes at a lower melting temperature. The primer and probe sequences for the H1N1 strain are localized to RNA Segment 4, a highly conserved area within a highly variable region of the hemagglutinin (HA) gene. The sequences additionally contain the substitution of particular bases with those observed in the 2009 H1N1 strains. Nucleotides “Y” and “R” are degenerative nucleotides. Further, the H1N1 primer and probe sequences do not cross-react with other contemporary circulating strains of H1 influenza.

The PCR component, which can be any component as discussed herein or can be, or can include, the prime mix, is present in an amount to sufficiently dissolve the extracted genomic nucleic acid material. Where the component is lyophilized, it may be necessary to reconstitute the material with added water or other suitable solvent before, with, or after combining the extracted, fixed, genomic material. The PCR vessel loaded with the genomic material is placed in the PCR machine for a prescribed period of time. For example, the assay time may take from about 30 minutes to 180 minutes, preferably about 45 minutes to 150 minutes. In a more preferred embodiment, the assay time is about 60 minutes to 120 minutes. In embodiments where the PCR component is the prime mix, detection preferably can be achieved within approximately 90 minutes from extraction. These times are intended to encompass preferred times for both DNA amplification, as well as RNA amplification that includes about 30-35 minutes for the reverse transcriptase step to convert the RNA to DNA. The exact time may be readily determined by those of ordinary skill in the art depending upon the material to be assayed and the type of PCR device selected.

The PCR reagents used in accordance with the present invention, in preferred embodiments, are designed to be at least substantially stable, and more preferably, stable. Specifically, the reagents in the form a prime mix of the present invention, are preferably substantially stable at room temperature, and this stability is measured and standardized as shown in FIG. 1A, FIG. 1B, FIG. 2A, FIG. 2B, and FIG. 2C. FIG. 1A and FIG. 1B illustrates the stability of the pathogen detection reagents. The standard chosen here is 1 picogram of initial cRNA from Flu A and H5 influenza. The Flu A and H5 samples were stored at −20° C., 4° C., and at room temperature (about 25° C.). Along the Y axis is the number of PCR cycles that have been run to register a reading. Stability as defined by the present invention is functional stability of the prime mix, which is indicated by detection of the sequence through amplification and fluorescence. In FIG. 1A and FIG. 1B, functional stability is indicated by detection of the sequence (with initial sample containing 1 pg of cRNA) up to 35 PCR cycles. The level of baseline fluorescence signifying a positive reading of a sample, and thus detection thereof, is referred to at the C_T value. The Cycle Threshold (C_T) is defined as the fractional cycle number at which the fluorescence passes the threshold. The threshold level is the Delta Rn used for C_T determination in real-time assays. The level is set to be above the baseline and sufficiently low so that it is within the exponential growth region of the amplification curve. The Delta Rn is the magnitude of the signal generated by the specified set of PCR conditions (Delta Rn=Rn−baseline). (See ABI Relative Quantification Users Guide for 7300/7500/7500 Fast Systems, Copyright 07.2006.)

As shown in FIG. 1A and FIG. 1B, stability is defined as the ability to reach C_T at 35 cycles or less, using 1 pg of cRNA in the starting sample. Thus, “substantially stable” encompasses prime mixes of the invention that are detectable at 1 pg through no more than 35 PCR cycles after the prime mix is stored at the specified temperature over time. For example, “substantially stable” includes prime mixes stored at room temperature for up to about 2 weeks, preferably up to about 4 weeks, and more preferably up to about 2 months or even about 3 months, where the prime mix is still useful and functioning for its intended purpose as measured by detection at 1 pg amounts in no more than 35 PCR cycles. The prime mixes are at least substantially stable for even longer periods of time at the various tested temperatures below room temperature.

FIG. 2A, FIG. 2B, and FIG. 2C show the results of a Pathogen Detection Reagent Stability Study, wherein the initial amount of cRNA is 1 femtogram. Here the cDNA is from Flu A, Flu B, and H5 influenza. FIG. 1A, FIG. 1B, FIG. 2A, FIG. 2B, and FIG. 2C illustrate the superior stability of a selection of prime mix reagents of the present invention at all three temperatures. As can be seen from the graph, the prime mix reagents for all three viruses remains stable at −20° C. and 4° C. to day 22. The stability profile at room temperature is also surprising, lasting about two weeks or more. It is further noted that the deviation around day 21 in FIG. 1A, FIG. 1B, FIG. 2A, FIG. 2B and FIG. 2C is likely due to the fact that the sample size was extremely small. As can be observed from the graph, the results of testing on day 22 showed a return to the detection level consistent with earlier testing days, indicating that stability beyond day 22 can reasonably be expected.

Based upon the data presented in FIG. 1A, FIG. 1B, FIG. 2A, FIG. 2B and FIG. 2C, substantial stability is achieved for extended period of time when the sample is kept frozen at about −20 to about 0° C., when the sample is stored above freezing such as in a refrigerator from above 0° C. to about 4° C., and when the sample is stored above refrigeration temperature to room temperature such as in the range of about 5° C. to about 27° C. In another embodiment, substantial stability can occur even at temperatures from above about 27° C. to 40° C. or about 27° C. to 50° C. Without being bound by theory, it is believed that the colder to temperature, the longer the sample will remain stable. For instance, the sample may be stable when frozen for 3-6 months, when refrigerated for 1-4 months and at room temperature for about one month.

Stability may preferably be achieved by a variety of means as in known in art. such as by lyophilizing the components. In one embodiment, stability may be enhanced by maintaining the lyophilized components of the invention below room temperature until the product is ready to be assembled, transported, or used. Based on the data from the stability studies as discussed above, stability can be achieved and maintained over a period of at least two months, more preferably three months.

This stability can be achieved by the lyophilization of all the PCR reagents before being loaded in the vessels included in the enclosure or before being packaged (e.g., foil or blister packs) in association with vessels. Lyophilization procedures and reagents are described in Das et al. as described above. It is noted, however, that the lyophilization of the reagents in the Das paper includes lyophilization of a master mix (nucleotides, buffer, and polymerase) only.

In accordance with one embodiment the present invention, the lyophilized reagents are disposed in the PCR-adapted vessels. Advantageously, including and using lyophilization for all PCR reagents can achieve one or more of the following: minimize efforts to assay and otherwise detect and identify organisms, particularly in difficult environments or under difficult conditions, increase assay reliability due to minimized or avoided degradation of the components, require less skilled operators of the diagnostic tool of the invention, and ensure greater durability of all components.

Stability may be achieved through any suitable method known by those of ordinary skill in the art. While lyophilization is one preferred embodiment to prepare the prime mix of the invention, the reagents can be encapsulated, e.g., in a liposome or paraffin bead, that dissociates in a PCR at typical operating temperatures, as will be readily determinable by those of ordinary skill in the art. As the PCR process is typically run at about 50° C., the liposome or bead can be designed to melt or dissolve at this temperature. Stability can also be achieved by providing all the components of the prime mix in liquid form in either a test tube, a 96-well plate, or a capillary vessel of plastic or glass. Those of ordinary skill in the art will envision other available methods to achieve the necessary stability of the prime mix of the invention based on the guidance provided herein.

Following the assaying of the genomic nucleic acid with the PCR instrument, primers, and/or probes, the results can be analyzed. Whether one or more primers and/or probes have associated with one or more organisms is generally known to those of ordinary skill in the art, based on chemical indicators, colors, and other observable results based on the reactions. For example, the association between the primers and/or probes with genetic material from the organism may be detected by fluorescence to facilitate detection of the biological organism. In certain embodiments of the present invention, the kit includes an analyzer to provide diagnostic information as to which probes, if any, provide positive results (e.g., a positive result is detection by of an organism by a probe specific for that organism). In some embodiments, the analyzer is incorporated with the PCR assay.

Following assay and identification, the diagnostic product of the invention optionally but preferably also includes the necessary pharmaceutical agent, along with pharmaceutically acceptable carrier, in the field to treat the disease or condition associated with the organism, or one or more symptoms associated therewith. Although such pharmaceutical compositions can be packaged with the product, preferably they are packaged separately from the product, particularly where the conventional pharmaceutical components are less stable than the lyophilized components. This permits pharmaceutical compositions with a longer shelf life to be included in the portable enclosure or with other modular components thereof just prior to use or transport to a field site or nearby storage site. Preferably, the diagnostic product includes a plurality of desired pharmaceutical compositions in doses in a number sufficient to prevent or treat one or more conditions caused by a selection of biological organisms depending on which organism is detected and identified. Preferably, these are formulated conventionally in a desired dosage form and strength, such as a tablet, capsule, patch, solution, lotion, or the like. Varying dosage strengths may be provided for certain types of pathogens, as appropriate. Indeed, the portable enclosure can be loaded with different components of any kind, such as active pharmaceutical ingredients, depending on the expected biological organisms or patient population one of ordinary skill in the art might encounter, based, for example, on field location, first responder reports, prior experience, or the like. If the patients are expected to be pediatric or geriatric, a larger portion of liquid formulations may be selected, by way of example.

The active pharmaceutical ingredient optionally, but preferably associated with the portable enclosure may include one or more vaccines, biologics, therapies, drugs, prophylactics, compositions (e.g., immunogenic), antidotes, treatments, cures, or any other medical item that is directed towards the treating or preventing of selected biological organisms. For example, if certain influenza strains are identified, the diagnostic tool may include Tamiflu® (Roche Pharmaceuticals Inc., New Jersey, USA) to treat the corresponding influenza infection. If certain bacteria are identified, the diagnostic tool may include certain antibiotics, such as azithromycin, effective in treating the corresponding bacterial infection. The dosages will, in any case, be present in a therapeutically or prophylactically effective amount.

In a prototype assay according to one embodiment of the present invention and where the microbe is a virus, RNA is extracted from a swab or tissue and added to the prime mix. Optionally, a control can be added to the mix. The control can be a positive control such as cRNA of the target microbe to allow a comparison to determine the identity of the sample, and/or a negative control, such as RNase-free water. The control(s) can also be run in a separate assay, either concurrently or sequentially. Next, the prime mix with the added RNA sequence to be identified is run on any suitable PCR instrument based on the guidance herein coupled with that known to those of ordinary skill in the art. Detection occurs within about 90 minutes from the time of extraction of the sample. In an assay using the prime mix, a very small number of copies of the microbial sequence are needed for identification to occur. FIG. 3 is a standard curve of concentrations from about 0.1 ng to 10 ag showing fluorescence readings of a sample having only 10 copies of a sequence in the 10 ag concentration. The gel in the upper left hand corner of FIG. 3 was run to verify the data shown fluorescence curves. In FIG. 3, influenza B is the sample being identified. As shown by the curve, 10 copies is sufficient for identification. It is further noted that, in accordance with the present invention, it is possible to identify a sequence with 5 or fewer copies of the sequence.

Methods of the present invention include detection of a microbial sequence including obtaining genomic material from a biological sample and assaying the genomic material (i.e., nucleic acid) by adding the sample material to the prime mix. In certain embodiments of the invention, the components of the prime mix may be present in liquid form in one or more laboratory vessels, such as a test tube, a 96-well plate, or a capillary of plastic or glass. The assay is preferably adapted for use with any PCR instrument, which may also include any fluorescence instrument commercially available or known in the art, for real time detection of the microbial sequence.

The prime mix of the present invention can also be used for disease surveillance. Disease susceptibility diagnostic assays can be augmentative genetic prime mix assays of the invention that provide additional medical information about one or more bacterial or viral pathogens, such as resistance markers resulting from known genetic polymorphisms/mutations that confer resistance to known drug treatment modalities.

For example, a growing number of influenza A (H3N2) isolates obtained from patients in the U.S. revealed that 92.3% contain a change at amino acid 31 (S31N) in the M2 gene known to be correlated with adamantane resistance (e.g., amantadine and rimantadine) and 2 of 8 influenza A (H1N1) strains contained the same mutation (Bright et al., JAMA, 2006). Adamantane resistance among influenza A (H₃N₂) and some H1N1 strains is highlights the clinical importance of having rapid (point of care) surveillance for antiviral resistance. In accordance with one embodiment of the invention, the prime mix can target resistance markers (e.g., by use of a probe) for neuraminidase inhibitors.

Identification of influenza to a specific strain and sub-strain can be determined by the following. It should be understood that any viral or bacterial agent may be targeted according to the invention, and that most references herein to influenza may be replaced with any other suitable viral or bacterial agent. First, an assay is run by the method outlined above to determine if an influenza pathogen is present in a sample. If an influenza infective is present in the sample, a second test can be run to determine if the virus is influenza strain A or B. If the organism is identified as influenza A, one or more additional tests can be performed to determine the subtypes, such as H1, H3, H5, etc. Preferably, this is all achieved using the same sample so that no additional samples are required from the patient or subject. Alternatively, a single test can be run with the patient sample and the primers, probes, enzymes, or any combination for multiple strains and subtypes.

There are several advantages that can be imparted by use of the prime mix of the present invention. First, when the prime mix is already assembled in a suitable container for insertion into a PCR device, it is unnecessary to store multiple individual reagents and excess laboratory equipment at a site or to carry it in the portable enclosure of the invention. This is particularly beneficial in areas where access to storage, refrigeration, transport, or any combination, is not readily available, and where mobility is desired (as it is no longer necessary to transport and store individual reagents). A further benefit of a pre-assembled prime mix, when used, is that the identification process is streamlined, requiring less time, less mixing and pipetting, less cleaning or recycling of containers, and less margin for error in measuring. The simplified process can reduce the opportunity for user error and contamination, and can advantageously expedite assay results.

Additionally, the prime mix can permit easy and rapid detection of a sample microbial sequence. Current detection methods for pathogens and disease resistance can take up to several days. In contrast, by using the prime mix of the present invention, detection can be achieved within about 90 minutes in one embodiment. As illustrated by FIG. 1A, FIG. 1B, FIG. 2A, FIG. 2B, FIG. 2C, FIG. 3, and FIG. 10, detection can be achieved using any standard PCR instrument and a small amount of the starting sequence. With a starting amount of 1 million copies of a sequence, detection can be achieved in fewer than 35 cycles. Improved detection allows identification of smaller samples. This is particularly advantageous in instances where it is desired to run multiple tests from a single sample, leaving more sample available for additional tests. This feature may also be beneficial in the event that a sample has been compromised, perhaps through transit or exposure, and less sample is intact for testing.

Surprisingly, the prime mix exhibits markedly increased stability, lasting several days or longer at room temperature. In another more preferred embodiment, the prime mix exhibits substantial stability for at least about two weeks, and up to about one month at room temperature. Stability for an extended period of time at room temperature (approximately 23° C.-27° C.) imparts great flexibility in use in both traditional and non-traditional environments. For example, the assay can be used more reliably in hospitals, doctor's offices, as well as in remote locations, such as in areas that have been subject to a bioterrorist attack, natural disaster, or at battlefield. The invention can be used at airports and border crossings to help minimize or prevent infected individuals from spreading disease.

Further, the prime mix can provide great flexibility and compatibility of use. The prime mix is adapted for use with several rRt PCR formats, and is available ready-to-use in reaction vessels for direct and immediate analysis. The prime mix can be used with any extraction or purification kit, and can include any standard master mix components (buffer, nucleotides, polymerase) available in the art as well as components described herein.

It is noted that the present invention encompasses the prime mix alone, existing independently from any apparatus or kit. Separate, additional embodiments of the present invention are directed to the use of the prime mix with other apparatuses, kits, etc.

In one aspect of the invention, there is provided a method for detecting the presence or absence of an influenza A-specific nucleic acid. In particular aspects, the invention provides a method and compositions for detecting the presence or absence of an influenza B-specific nucleic acid. In some embodiments, there is provided a method for detecting the presence or absence of a particular subtype of influenza A, such as, for example, an H1N1 subtype, including, for example, such H1N1 strains that are commonly referred to as “swine flu” isolates.

In particular, the invention provides novel probes and primers that may be used to specifically detect the presence or absence of particular H1N1 subtype viral strains that are causal agents for the influenza viral strains that are the causal agents of the 2009 H1N1 swine flu.

In an overall sense, the invention provides methods and compositions for detecting an H1N1 subtype viral nucleic acid from within a plurality of polynucleotides obtained from a biological sample. There is also a method for quantitating the amount of H1N1 subtype viral nucleic acid within the sample and monitoring the efficacy of the formulations at stabilizing and protecting the molecular fidelity of the isolated polynucleotides.

In another aspect, the present invention provides a method for rapidly detecting in a biological sample, a particular polynucleotide sequence, such as an H1N1 Influenza A subtype-specific nucleic acid. In an overall and general sense, this method includes amplification of a population of nucleotides suspected of containing the particular sequence using conventional methods such as PCR and forward and reverse primers that are specific for the target sequence, hybridization of a specific probe set with the resulting single-stranded PCR product, performing melting curve analysis and analyzing the T_m change of the hybrid of the single-stranded PCR product with the hybridization probes.

In one embodiment, the present invention provides a method for rapidly detecting the presence of a polynucleotide (such as an H1N1 subtype viral nucleic acid sequence), using a PCR-based methodology, which generally includes: (a) isolating polynucleotides from the sample to be analyzed; (b) amplifying the polynucleotides by PCR using a primer set that is specific to the H1N1 subtype viral nucleic acid target sequence; (c) hybridizing one or more labeled probes that are specific for the polynucleotide of interest with the single-stranded PCR product obtained from step (b); and (d) detecting the presence of the labeled probe in the sample, indicative of the presence of the specific H1N1 subtype viral nucleic acid target sequence within the population of isolated polynucleotides.

In another aspect, the present invention provides a method for rapidly detecting in a biological sample, a particular nucleic acid sequence from among a population of polynucleotides isolated from a biological sample. In an overall and general sense, this method includes amplification of a population of nucleotides suspected of containing the particular sequence using conventional methods such as PCR and forward and reverse primers that are specific for the target sequence, hybridization of a specific probe set with the resulting single-stranded PCR product, performing melting curve analysis and analyzing the T_m change of the hybrid of the single-stranded PCR product with the hybridization probes.

One such method for the detection of polynucleotides using a labeled “probe” sequence utilizes the process of fluorescence resonance energy transfer (FRET). Exemplary FRET detection methodologies often involve pairs of fluorophores including a donor fluorophore and acceptor fluorophore, wherein the donor fluorophore is capable of transferring resonance energy to the acceptor fluorophore. In exemplary FRET assays, the absorption spectrum of the donor fluorophore does not substantially overlap the absorption spectrum of the acceptor fluorophore. As used herein, “a donor oligonucleotide probe” refers to an oligonucleotide that is labeled with a donor fluorophore of a fluorescent resonance energy transfer pair. As used herein, “an acceptor oligonucleotide probe” refers to an oligonucleotide that is labeled with an acceptor fluorophore of a fluorescent resonance energy transfer pair. As used herein, a “FRET oligonucleotide pair” will typically include an “anchor” or “donor” oligonucleotide probe and an “acceptor” or “sensor” oligonucleotide probe, and such pair forms a FRET relationship when the donor oligonucleotide probe and the acceptor oligonucleotide probe are both hybridized to their complementary target nucleic acid sequences. Acceptable fluorophore pairs for use as fluorescent resonance energy transfer pairs are well known to those skilled in the art and include, but are not limited to, fluorescein/rhodamine, phycoerythrin/Cy7, fluorescein/Cy5, fluorescein/Cy5.5, fluorescein/LC Red 640, and fluorescein/LC Red 705, and the like.

Primers useful in amplification of a particular sequence of interest may be designed using, for example, a computer program such as OLIGO® (Molecular Biology Insights Inc., Cascade, Colo., USA). Typically, oligonucleotide primers are from about 10 to about 60 or so nucleotides in length (including, without limitation, all intermediate integers, e.g., 10, 11, 12, etc., or even 60 or more nucleotides in length), although primers of any practical length may be useful in the practice of certain embodiments of the invention.

The invention also provides a method for increasing the efficiency of obtaining a purified population of H1N1 subtype viral nucleic acids from a biological sample suspected of containing such H1N1-specific nucleic acids. In an overall and general sense, the method includes contacting the sample with a composition that includes one or more of the formulations described herein, in an amount and for a time sufficient to increase the efficiency of obtaining the purified population of H1N1-specific polynucleotides from the biological sample.

In exemplary embodiments, the integrity of a population of polynucleotides in the biological sample, and/or the fidelity of at least a first sequence of at least one of the polynucleotides obtained from the sample is at least substantially maintained (i.e., at least 75% of the nucleotides within the population are substantially full-length) when the composition including the sample is stored at a temperature of from about −20° C. to about 40° C. for a period of from about 7 to about 14 days or longer; alternatively at a temperature of from about −20° C. to about 40° C. for a period of from about 7 to about 14 days or longer; or alternatively at a temperature of from about 20° C. to about 40° C. for a period of from about 14 to about 30 days or more.

Alternatively, the integrity of a population of polynucleotides in the biological sample is at least substantially maintained such that at least about 80%, at least about 85%, at least about 90%, or at least about 95% or more of the nucleotides within the population are present in the solution when compared to the amount present in the solution when the sample was initially collected. In preferred embodiments, the integrity of the sample will be substantially maintained such that all, or almost all of the viral-specific polynucleotides present in the initial sample will be maintained (i.e., not detectably degraded) over time.

1. A method for detecting the presence or absence of an Influenza A H1N1 subtype virus-specific nucleic acid segment in a population of polynucleotides in a sample, which method comprises: (a) performing at least one cycling step, wherein the cycling comprises at least a first amplifying step and at least a first hybridizing step, wherein the at least a first amplifying step comprises contacting the sample with a pair of different, independently selected Influenza A H1N1 subtype virus-specific amplification primers to produce an Influenza A H1N1 subtype virus-specific amplification product if an Influenza A H1N1 subtype virus-specific nucleic acid segment is present in the sample; and (b) detecting the presence of the amplification product using a labeled oligonucleotide probe that is specific for the H1N1 amplification product, wherein the presence of the amplification product is indicative of the presence of one or more Influenza A H1N1-specific nucleic acid segments in the population of polynucleotides.

2. The method of claim 1, wherein the pair of amplification primers comprises a first oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence AGCCTYCCATTTCAGAATATACA (SEQ ID NO:51).

3. The method of claim 2, wherein the pair of amplification primers comprises a first oligonucleotide primer of less than about 40 nucleotides in length that comprises the nucleic acid sequence AGCCTYCCATTTCAGAATATACA (SEQ ID NO:51).

4. The method of claim 3, wherein the pair of amplification primers comprises a first oligonucleotide primer that consists essentially of the nucleic acid sequence AGCCTYCCATTTCAGAATATACA (SEQ ID NO:51).

5. The method of claim 1, wherein the pair of amplification primers comprises a second oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence AATCCTGTRGCCAGTCTCAATTTTG (SEQ ID NO:52).

6. The method of claim 5, wherein the pair of amplification primers comprises a second oligonucleotide primer of less than about 40 nucleotides in length that comprises the nucleic acid sequence AATCCTGTRGCCAGTCTCAATTTTG (SEQ ID NO:52).

7. The method of claim 1, wherein the pair of amplification primers comprises a first oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO:51, and a second oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO:52.

8. The method of claim 7, wherein the pair of amplification primers comprises a first oligonucleotide primer of less than about 40 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO:51, and a second oligonucleotide primer of less than about 40 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO:52.

9. The method of claim 1, wherein the detection probe comprises a first oligonucleotide probe of less than about 50 nucleotides in length, and further wherein the first oligonucleotide probe comprises the nucleic acid sequence of TCCAAAATATGTAAAAAG (SEQ ID NO:53).

10. The method of claim 9, wherein the detection probe comprises a first oligonucleotide probe of less than about 30 nucleotides in length, and further wherein the first oligonucleotide probe comprises the nucleic acid sequence TCCAAAATATGTAAAAAG (SEQ ID NO:53).

11. The method of claim 1, wherein (a) the pair of amplification primers comprises: (i) a first oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO:51, and (ii) a second oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO:52; and (b) the detection probe comprises a first oligonucleotide probe of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO:53.

12. The method of claim 11, wherein (a) the pair of amplification primers comprises: (i) a first oligonucleotide primer that consists essentially of the nucleic acid sequence of SEQ ID NO:51, and (ii) a second oligonucleotide primer that consists essentially of the nucleic acid sequence of SEQ ID NO:52; and (b) the detection probe comprises a first oligonucleotide probe that consists essentially of the nucleic acid sequence of SEQ ID NO:53.

13. The method of claim 1, wherein the biological sample is selected from the group consisting of blood, plasma, cells, tissues, and serum.

14. An Influenza A H1N1 Virus-specific oligonucleotide amplification primer set, wherein the first amplification primer is less than about 50 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO:51 and the second amplification primer is less than about 50 nucleotides in length and comprises the nucleotide sequence of SEQ ID NO:52.

15. The Influenza A H1N1 Virus-specific oligonucleotide amplification set of claim 14, further comprising a first detection probe, wherein the first detection probe comprises a labeled oligonucleotide probe of less than about 50 nucleotides in length that comprises the nucleotide sequence of SEQ ID NO:53.

16. A kit comprising, in suitable container means, the Influenza A H1N1 Virus-specific oligonucleotide amplification primer set of claim 14, and instructions for using the primer set in a PCR amplification of a population of polynucleotides obtained from a biological sample or specimen.

17. The kit of claim 16, further comprising, in suitable container means, an oligonucleotide detection probe that specifically binds to the amplification product produced from PCR amplification of a population of polynucleotides obtained from a biological sample or specimen that contains, or is suspected of containing, an Influenza A H1N1 Virus-specific nucleic acid segment.

18. An article of manufacture, comprising: a pair of Influenza A H1N1 Virus-specific oligonucleotide amplification primers; and a first Influenza A H1N1 Virus-specific oligonucleotide detection probe; wherein the detection probe comprises at least one detectable label.

19. The article of manufacture of claim 18, further comprising a package insert having instructions for using the pair of primers and the detection probe to detect the presence or absence of an Influenza A H1N1 Virus-specific nucleic acid segment within a population of polynucleotides obtained from a biological sample that was collected from a human subject.

20. A composition comprising: (a) a first pair of Influenza A H1N1 Virus-specific amplification primers, wherein the pair of primers comprises: (i) a first oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO:51; and (ii) a second oligonucleotide primer of less than about 50 nucleotides in length that comprises the nucleic acid sequence of SEQ ID NO:52; and (b) a first Influenza A H1N1 Virus-specific oligonucleotide detection probe, comprising: (i) a first oligonucleotide detection probe of less than about 50 nucleotides in length, wherein the probe comprises the nucleic acid sequence of SEQ ID NO:53; and (ii) at least a first detection reagent operably linked to the oligonucleotide detection probe.

21. The composition of claim 20, wherein the polynucleotide is amplified from a population of polynucleotides obtained from a biological sample.

22. The composition of claim 21, wherein the polynucleotide is amplified from a population of polynucleotides obtained from a human suspected of infection by an H1N1 subtype of Influenza A Virus.

23. The composition of claim 20, wherein the oligonucleotide detection probe comprises a radioactive, luminescent, chemiluminescent, fluorescent, enzymatic, magnetic, or spin-resonance label.

24. The composition of claim 20, further comprising one or more components, buffers, enzymes, or reagents for performing polymerase chain reaction.

25. The composition of claim 20, wherein the composition is at least substantially stable at room temperature and is adapted and configured for use with a polymerase chain reaction (PCR) device.

26. A biological organism identification product that comprises: (a) a collection device to collect one or more sample organisms; (b) a fixing and transporting composition present in an amount sufficient to kill the one or more sample organisms associated with the collection device; (c) an extraction member to extract a sufficient amount of genomic nucleic acid from the one or more sample organisms to facilitate identification thereof; and (d) a substantially stable polymerase chain reaction (PCR) component into which the sufficient amount of genomic nucleic acid can be added, and further wherein the PCR component comprises the composition of claim 30.

27. A method of identifying a subtype of Influenza A virus comprising: detecting the presence of an Influenza A virus-specific nucleic acid segment, if present, in a population of polynucleotides using a labeled oligonucleotide probe that is specific for the Influenza A virus-specific nucleic acid segment; and if an Influenza A virus-specific nucleic acid segment is present in the population of polynucleotides, then further identifying particular subtype of Influenza A virus using a labeled oligonucleotide probe that is specific for an H5 or an H1N1 subtype of Influenza A virus.

28. The method of claim 27, wherein the further identifying is performed by contacting the nucleic acid segment with a labeled oligonucleotide probe that is specific for an Influenza A H5 subtype, or an Influenza A H1N1 (Swine) subtype, and detecting the presence of the labeled hybridization product so produced.

29. The method of claim 28, wherein the detection of the Influenza A H5 subtype and the Influenza A H1N1 (Swine) subtype is sequential.

30. The method of claim 29, wherein the labeled oligonucleotide probe comprises a nucleic acid sequence selected from the group consisting of 5′-TCCAAAATATGTAAAAAG-3′ (SEQ ID NO:53), 5′-TCAGGCCCCCTCAAAGC-3′ (SEQ ID NO:54); 5′-ATGGGAAATTCAGCTCT-3′ (SEQ ID NO:55); 5′-TCTCCAAAGTATGTCAGG-3′ (SEQ ID NO:56); 5′-TGAGATCAGATGCACCCAT-3′ (SEQ ID NO:57); 5′-AGAGRGGAAATAAGTGG-3′ (SEQ ID NO:58), and any combination thereof.

31. The method of claim 30, wherein the oligonucleotide probe is labeled at its 5′-end with FAM.