WO2015183811A1

WO2015183811A1 - Apparatus and methods for detecting and identifying nucleic acid sequences in biological samples

Info

Publication number: WO2015183811A1
Application number: PCT/US2015/032432
Authority: WO
Inventors: Gerald W. Fischer; Luke T. Daum
Original assignee: Longhorn Vaccines And Diagnostics, Llc
Priority date: 2014-05-28
Filing date: 2015-05-26
Publication date: 2015-12-03

Abstract

The invention is directed to an extraction apparatus and methods to extract nucleic acid from a sample, and preferably to isolate, quantify and identify target sequences. The extraction system contains a sterile vessel to efficiently isolate only the target sequence that possesses a tapered end containing nucleic acid absorbable materials such as silicon dioxide. The invention is directed to kits, compositions, tools and methods for isolating, detecting, amplifying, and quantitating pathogen-specific nucleic acids in a biological sample. The invention provides diagnostic kits containing specific amplification primers, and labeled detection probes that specifically bind to the amplification products. Also disclosed are compositions and methods for the isolation and characterization of nucleic acids that are specific to one or more pathogens, including for example Influenza virus and Mycobacterium tuberculosis, from a wide variety of samples including those of biological, environmental, clinical and/or veterinary origin.

Description

APPARATUS AND METHODS FOR DETECTING AND IDENTIFYING NUCLEIC ACID SEQUENCES IN BIOLOGICAL SAMPLES

Background

1. Field of the Invention

The present invention generally relates to systems, kits, apparatuses, tools, compositions and methods for detecting, quantitating and identifying, nucleic acid sequences within a biological sample. In particular, the systems, kits and methods of the invention provide for the safe, efficient and rapid extraction of target nucleic acid sequences for PCR amplification.

2. Background of the Invention

Conventional diagnosis of an infection of Mycobacteria tuberculosis (TB) relies on a combination of physical examination (e.g., chronic persistent cough, enlarged or tender lymph nodes, pleural effusion, unusual breath sounds, and, in later stages of the disease, characteristic "clubbing" of the fingers or toes) and diagnostic testing (e.g., sputum examination, microbial culture and nucleic acid testing of specimens, bronchoscopy, CT scan or X-ray of the chest, pulmonary biopsy, thoracentesis, interferon-γ (gamma) blood test, and tuberculin skin test).

The "standard" of TB diagnostics, cell culturing of mycobacterial organisms, is difficult, due in part to their long generation times, i.e., twenty-four hours for M. tuberculosis. In addition, mycobacteria are typically present at low levels in infected individuals. Culturing from a clinical specimen can therefore take anywhere between four to eight weeks, during which time a patient may become seriously ill and contagious to others. In addition, cell culturing requires the collection, transport and maintenance of viable mycobacterial organisms in a sample until such time as the sample can be analyzed in a lab setting. In countries where TB is prevalent, and health care is minimal, this may not be an option, thus increasing the risk of spreading infection.

For regions with limited access to medical care, the whole blood must be analyzed within 12 hours of obtaining the sample. Effectiveness of the test has not been analyzed on patients with other medical conditions such as HIV, AIDS, diabetes, silicosis, chronic renal failure, hematological disorders, and individuals treated for TB infection. Nor has effectiveness been tested on pregnant individuals or minors ("Clinicians Guide to QuantiFERON® -TB Gold," Cellestis). Other non-culture methods such as radioimmunoassays, latex agglutination, and enzyme-linked immunosorbent assays (ELISAs) have been used with limited degrees of success to confirm the presence of tubercle bacilli in biological samples.

The majority of clinical diagnostic laboratories employed traditional culture for pathogen identification that typically requires 3-7 days for most viruses and longer for some bacterial strains, including up to about 21 days for the culturing of M. tuberculosis. Traditional culture requires specimen collection of viable microbes, frozen transport, and propagation and handling of potentially infectious and often unknown biological microbes. Furthermore, many infectious agents, e.g., highly pathogenic avian influenza, SARS, M. tuberculosis complex, etc., are BSL-3 level pathogens that require specialized facilities and precautions for analysis. There are challenges in obtaining, shipping and maintaining high-quality, and viable biological specimens for culture. Specimens must be shipped using a cold chain, most often dry ice. Transporting potentially infectious samples from remote sites or across international borders using commercial transit can be costly and tedious, particularly when specimens must be received frozen.

Collection is the first step in diagnostic platforms or molecular protocols requiring the detection of potentially minute amounts of nucleic acids from microbes. Regardless of the nucleic acid test used or the RNA/DNA extraction protocol, specimen collection, specifically the inactivation of potentially infectious agents and the preservation and stability of pathogen RNA/DNA remains a critical gap in clinical diagnostics, especially for use around the world.

Typically, patients suspected of having tuberculosis are asked to cough hard and then expectorate into a specimen cup in order to obtain a sputum sample. Usually, this procedure is done in a well ventilated area so as to minimize the potential for spreading infective mycobacteria. Patients may be asked to repeat this procedure in order to collect enough sputum for analysis, typically in amounts from about 5 mL to about 20 mL. Typically, collected sputum samples are refrigerated until further analytic procedures, such as cell culturing or decontamination procedures to inactivate or kill any microorganisms contained within the sample, can be performed. To detect Mycobacterium tuberculosis in a sputum sample, an excess of 10,000 organisms per mL of sputum are needed to visualize the bacilli with a 100X microscope objective (1000X magnification). Direct smear microscopy of sputum samples from tuberculosis patients is typically regarded as an effective tool for monitoring patient response to treatment. Typically, more acid fast bacilli will be found in the purulent portions of the sputum.

The majority of current diagnostic laboratories have transitioned from traditional culture to nucleic acid testing (NAT) such as real-time PCR. Nucleic acid amplification testing for TB includes the use of standard polymerase chain reaction (PCR) techniques to detect mycobacterial DNA in patient specimens, nucleic acid probes to identify mycobacteria in culture, restriction fragment length polymorphism (RFLP) analysis to compare different strains of TB for epidemiological studies, and genetic -based susceptibility testing to identify drug-resistant strains of mycobacteria. Real-time PCR (RT-PCR) and real-time reverse transcription PCR (rRT— PCR) are significant improvements and provide superior sensitivity and specificity results in hours.

The complete genome of M. tuberculosis has been sequenced and published; currently two nucleic acid amplification-based tests for TB have been approved for use in the United States by the Food and Drug Administration (FDA). The first, known as the "Enhanced Amplified Mycobacterium Tuberculosis Direct Test" (E-MTD, Gen-Probe, San Diego, CA, USA), is approved for detection of M. tuberculosis complex bacteria in acid-fast bacilli in both smear-positive and smear-negative respiratory specimens from patients suspected of having TB. The E-MTD test combines isothermal transcription-mediated amplification of a portion of the 16S rRNA with a detection method that uses a hybridization probe specific for M. tuberculosis complex bacteria. The second, known as the AMPLICOR® Mycobacterium tuberculosis Test (AMPLICOR®, Roche Diagnostics, Basel, Switzerland), has been approved for the detection of M. tuberculosis complex bacteria only in smear-positive respiratory specimens from patients suspected of having TB. This test uses PCR to amplify a portion of the 16S rRNA gene that contains a sequence that hybridizes with an oligonucleotide probe specific for M. tuberculosis complex bacteria. ("Report of an Expert Consultation on the Uses of Nucleic Acid Amplification Tests for the Diagnosis of Tuberculosis," Centers for Disease Control and Prevention).

Results have indicated that the sensitivity and specificity of these tests tends to vary depending on geographical location and risk factors. In addition, these techniques require complex laboratory conditions and equipment to be performed, thus reducing the speed and sensitivity of the test. For these and other reasons, there remains a need in the art for reliable and accurate methods for detection of Mycobacterial pathogens and other microbes in clinical samples, and in particular, methods for rapidly identifying such pathogens in field applications, remote locations, and in developing countries where conventional laboratories are lacking, and financial resources are limited. There is also a need for new tools and methods whereby the microbe is killed and lysed to facilitate extraction efficiency and eliminate biohazard risk for easy disposal. In particular, kits for the safe collection, handling, and transport of pathogenic specimens, as well as molecular biology-based methods for efficient extraction, rapid detection and accurate identification of microbial nucleic acids to include TB-specific nucleic acids in such specimens are highly desired. Summary of the Invention

The present invention overcomes the problems and disadvantages associated with current strategies and designs, and provides kits, apparatuses, compositions, tools and methods for detecting and identifying target nucleic acid sequences and genomes.

One embodiment of the invention is directed to an extraction system for simply and efficiently, as compared to conventional systems, extracting a target nucleic acid from a biological sample comprising: a sterile vessel tapered at one end with no O ring, wherein the tapered end contains an agent that binds nucleic acid; a sterile lysis buffer that, when added to the biological sample, sterilizes the sample and denatures nucleic acid of the sample; at least one sterile wash buffer that does not disrupt binding of nucleic acid to the agent; and an elution buffer that promotes release of nucleic acid from the agent. Preferably the target nucleic acid is specific for a pathogen such as, for example, a bacterium, a virus, or a parasitic or fungal microorganism. Preferred pathogens include the causative agents of cholera, tuberculosis, influenza, SARS, MERS, HIV, AIDS, malaria or measles. Preferably the biological sample is a sample of fluid or tissue from a person suspecting of being infected by or at risk of infection from the pathogen. Preferably the lysis buffer contains a chaotrope, an anionic detergent, a reducing agent, a chelator, a surfactant or antifoaming agent, nuclease-free water, a pH buffer, and no enzymes and the lysis buffer and/or the at least one wash buffer contains a control nucleic, which improves the efficiency of extraction of nucleic acid and/or serves as a positive control of extraction efficiency. Preferably the at least one wash buffer contains a salt and a buffering agent, the elution buffer contains a salt and a buffering agent, and the agent is a silica dioxide wafer. The invention may further comprise a sterile alcohol, a testing buffer that contains an osmolarity agent, a chelator, magnesium ions, a dye, a mixture of deoxynucleotide triphosphates, a pH buffer, primers pairs specific for PCR amplification of the sequence of the target nucleic acid and optionally a heat-stable polymerase. The invention may further comprise a sterile device for collection of the biological sample and a sterile vessel for transporting the biological sample collected, preferably wherein the sterile vessel contains lysis buffer that sterilizes the biological sample.

Another embodiment of the invention is directed to methods comprising: adding a biological sample containing a target sequence to a sterile vessel containing a lysis buffer forming a sterile mixture; adding the sterile mixture to a sterile cylindrical vessel tapered at one end and without an O ring, wherein the tapered end contains an agent that binds to nucleic acid; subjecting the sterile mixture to centrifugation so that the mixture passes through the agent and nucleic acid of the mixture binds to the agent; contacting the agent with at least one wash buffer that does not interfere with binding of nucleic acid to the agent; and contacting the agent with an elution buffer that causes a release of nucleic acid from the agent such that nucleic acid collects in the elution buffer forming an elution mixture. Preferably the biological sample is fluid or tissue from a person suspecting of or at risk of infection by a pathogen, the target sequence is specific to nucleic acid of the pathogen and the pathogen is a bacterium, a virus, or a parasitic or fungal microorganism such as, for example, a pathogen that is the causative agent of one or more of cholera, tuberculosis, influenza, SARS, MERS, HIV, AIDS, malaria or measles. Preferably the lysis buffer contains a chao trope, an anionic detergent, a reducing agent, a chelator, a surfactant or antifoaming agent, nuc lease-free water, a pH buffer, and no enzymes and the lysis buffer and/or the at least one wash buffer contains a control nucleic that improves extraction efficiency of nucleic acid and/or serves as a positive control of extraction efficiency. Preferably the at least one wash buffer contains a salt and a buffering agent, the elution buffer contains a salt and a buffering agent and the agent is a silica dioxide wafer. Preferably, the invention further comprises the option of adding sterile alcohol to the biological sample prior to centrifugation and does not require an incubation step. Preferably the method further comprise adding a testing buffer to the elution mixture, wherein the testing buffer contains an osmolality agent, a chelator, magnesium ions, a dye, a mixture of deoxynucleotide triphosphates, a pH buffer, primers pairs specific for PCR amplification of the sequence of the target nucleic acid and a heat-stable polymerase and, the target sequence is amplified, quantitated and sequenced by PCR analysis. Preferably the amplified target sequence has a Ct value that is less than the Ct value of the amplified target sequence obtained from conventional PCR of a biological sample and, also preferably, the Ct value is 10% lower than the Ct value of the amplified sequence obtained from conventional PCR of a biological sample.

Another embodiment of the invention is directed to an apparatus comprising: a sterile vessel cylindrical in shape and configured for addition of a biological sample at one open end and tapered at another open end, wherein the tapered end contains an agent that binds a macromolecule. Preferably the vessel contains no O ring, is cylindrical and configured for centrifugation such that upon centrifugation the biological sample would move towards the agent. Preferably the inner walls of the vessel toward the tapered end are smooth with no ridges. Also preferably, the agent is a silica dioxide wafer, larger than the another open end, and the macromolecule is a nucleic acid. Preferably the apparatus is a spin-column for extracting nucleic acid from a biological sample. Other embodiments and advantages of the invention are set forth in part in the description, which follows, and in part, may be obvious from this description, or may be learned from the practice of the invention.

Description of the Drawings

Figure 1 Detection of Mycobacterium tuberculosis DNA using PrimeXtract or Qiagen Spin-Columns, each with QiaAmp DNA mini kit reagents and protocol.

Figure 2 Detection of Mycobacterium tuberculosis DNA using PrimeXtract or Qiagen Spin-Columns, each with PrimeXtract lysis and wash buffers.

Figure 3 Detection of Mycobacterium tuberculosis DNA using PrimeXtract and PrimeXtract lysis and was buffers and protocol, or Qiagen Spin-Columns with QiaAmp DNA mini kit reagents and protocol.

Figure 4 Depiction of Conventional spin-column (prior art).

Figure 5 Depiction of PrimeXtract spin-column.

Description of the Invention

It has been surprisingly discovered that a target nucleic acid sequences can be reliably and accurately detected, and with increased sensitivity as compared to conventional systems, in samples such as biological samples with minimal equipment and supplies and minimal and/or no special or unique training of personnel. The systems, kits, apparatus and methods of the invention allow for rapidly identifying pathogen-specific target sequences in the laboratory, in field applications, in remote locations, and in developing countries where conventional laboratories and financial resources are limited, and where the sample is sterilized to eliminate biohazard risk and for safe disposal of materials and supplies. These safe and highly efficient system, kits and methods allow for the straightforward collection, handling and transport of biological samples suspected of containing pathogenic organisms. In particular, systems, kits and methods of the invention provide for the specific detection of one or more strains of pathogenic microorganisms such as bacteria, viruses, yeast, fungus and/or parasites. In particular applications, the invention encompasses a diagnostic product that permits the collection of a target specimen, preparation of the target specimen for assaying, isolation of genomic material from the specimen, and subsequent processing of the genomic material to identify one or more organisms, if present, in the biological sample. When coupled with one or more specimen collection devices, the compositions of the invention permit safe, collection, transport and storage of biological specimens, even for those collected in remote or field applications, wherein the time from sample collection to sample assay may be hours to days, or even weeks.

The invention further encompasses compositions and methods that simplify and expedite specimen collection, nucleic acid extraction, preparation and molecular detection of microorganisms such as, for example, those microorganisms that are the causative agents of viral, bacterial, parasitic or fungal diseases such as influenza and tuberculosis. In particular applications, the invention encompasses a diagnostic product whereby the specimen is collected, transported and rapidly prepared for downstream PCR without the need for refrigeration or costly and time-consuming sample decontamination and specimen emulsification. The invention also encompasses systems and methods for assessing and following epidemiologic and outbreaks and surveillance of pandemic and epidemic tracking and microbial sequencing directly from field samples at the site of collection or by using inexpensive, simplified, safe systems. The systems and methods of the invention also includes tools and method that are easily amenable to further diagnostic analysis such as quantitation and sequencing of nucleic acids of interest.

The systems and methods of the invention provide pathogen-specific or pathogen nonspecific nucleic acid detection. Probes and amplification primers specific to pathogens of interest are incorporated into the systems and methods of the invention. The invention also provides facile identification of pathogens in collected samples, and permits a safe, cost- effective, and near-term assessment of infection, including, for example, as a tool in surveillance against potential epidemics, monitoring of outbreaks, assessment of disease progression in affected or at-risk populations, and/or identification of particular species and/or strains of the microorganism for diagnostic testing or determining particular therapeutic modalities.

In one embodiment, the invention provides systems and methods for extracting nucleic acid from a sample, such as, for example, a biological, environmental or other sample that is known or suspected to contain nucleic acid. Preferably the sample is a biological sample suspected of containing one or more pathogenic and/or infectious organisms and microorganisms, (collectively the "pathogens"). In an overall sense, these systems and methods generally involve contacting a sample suspected of containing one or more pathogens with a collection buffer for an effective amount of time and with a sufficient amount of a composition that includes: a) one or more chao tropes; b) one or more detergents; c) one or more reducing agents; d) one or more chelators; and e) one or more surfactants, to kill substantially all, and preferably to kill all of the pathogenic organisms therein, including, for example, pathogenic bacteria, fungi, and viruses (if present in the sample). In the practice of the method, substantially all (and preferably, all) of the cells and microorganisms contained therein are lysed, and their cellular contents liberated into the solution. Preferably, substantially all (and more preferably, all) of the cellular enzymes, proteins, peptides, lipoproteins, and other cellular contents are denatured and/or inactivated, including any exogenous or endogenous nucleases that may be present in the sample, such that the resulting mixture is rendered substantially safe (and preferably, safe) for handling, storage, and/or transport by workers without undue effects, and without the need for concern over pathogenicity, toxicity, or danger of handling the sample now that it has been decontaminated and any pathogenic organisms originally present therein, destroyed, inactivated, killed, and/or lysed to render them harmless. Compositions for the collection of biological samples may be maintained in ready-to-use concentrations, or in concentrated forms such as, for example, 2x, 5x, lOx, 20x 25x, 30x, or greater as convenient or necessary for the particular application.

Preferably the nucleic acids of the biological sample collected are stable, such that the nucleic acid sequences do not degrade and the integrity of the nucleic acids are preferably substantially maintained, so that the obtained nucleic acids are intact, and present in the sample in the form that they were in when the cells containing them were initially liberated/lysed by the action of the components present in a composition of the invention. In preferred applications of the invention, the population of pathogen-specific polynucleotides obtained using the disclosed methods are substantially stable and non-degraded such that they can be maintained for significant periods of time even at less-than-ideal temperatures such as ambient and other temperatures (e.g., collection temperatures of about 0°C to even about 40°C or more) for extended periods of time (e.g., for periods of several hours to several days to several week or months even) without significantly degrading the liberated nucleic acids, thereby making them suitable for downstream molecular analysis with the same or nearly the same sensitivity of detection (e.g., template-dependent amplification reactions et al.) days to weeks after extraction of the nucleic acids takes place, even when it is not possible to store the populations of polynucleotides extracted from the samples frozen, on ice, or refrigerated between initial sample collection and subsequent molecular analysis.

The (i) the one or more chaotropes preferably include guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride, or any combination thereof; (ii) the one or more detergents preferably include sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, N-lauroyl sarcosine, or any combination thereof; (iii) the one or more reducing agents preferably include 2-mercaptoethanol, tris(2-carboxyethyl) phosphine, dithiothreitol, dimethylsulfoxide, or any combination thereof; (iv) the one or more chelators preferably include ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine pentaacetic acid, NN-bis(carboxymethyl)glycine, ethylenediaminetetraacetic, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof; or (v) the one or more buffers preferably include tris(hydroxymethyl) aminomethane, citrate, 2-(N-morpholino)ethanesulfonic acid, NN-Bis(2-hydroxyethyl)-2- aminoethanesulfonic acid, l,3-bis(tris(hydroxymethyl)methyl amino)propane, 4-(2- hydroxyethyl)-l-piperazine ethanesulfonic acid, 3-(N-morpholino) propanesulfonic acid, bicarbonate, phosphate, or any combination thereof.

Preferred formulations that are at ready-to-use concentrations include: (a)(i) about 3 M guanidine thiocyanate; (ii) about 1 mM TCEP; (iii) about 10 mM sodium citrate; (iv) about 0.5% N-lauroyl sarcosine; (v) about 0.0002% silicone polymer; (vi) about 100 mM 2-amino-2- hydroxymethyl-propane-l,3-diol (TRIS); and (vii) about 0.1 mM EDTA; or (b) (i) about 3 M guanidine thiocyanate; (ii) 1 mM TCEP; about 10 mM sodium citrate; (iii) about 0.5% N-lauroyl sarcosine, sodium salt; (iv) about 0.0002% of a silicone polymer; (v) about 100 mM TRIS; (vi) about 0.1 mM EDTA; and (vii) about 10% to about 25% ethanol (vol./vol.).

Because of the remarkable effectiveness of the disclosed formulations in readily killing, and lysing the cells and cellular components, denaturing the proteinaceous cellular components and inactivating enzymes such as endogenous and exogenous nucleases that are deleterious to the preservation of intact nucleic acids, the inventors have demonstrated that in certain instances, substantially all of the microorganisms present in a sample are killed and/or lysed within the first few minutes it is contacted with the composition. In some instances, the killing and lysing of the cells is substantially complete within about 3 or about 4 or about 5 or so minutes of contacting the sample with the composition. Likewise, in other instances, contacting the sample with the composition for a period of about 6, or about 7, or about 8, or about 9, or about 10 minutes or so is sufficient to substantially kill and/or lyse all of the pathogens that may be present in the collected sample. Likewise, substantially all of the proteins, enzymes, nucleases, and the like liberated from the lysed cells present in a sample are substantially all inactivated and/or denatured within only a few minutes of contacting the sample with the composition. Preferably the samples are of biological, clinical, veterinary, or environmental origin, and in certain embodiments, the samples are preferably of human origin, and in particular, from humans that have, are suspected of having, or are at risk for developing a microbial infection, such as a tubercular infection caused by one or more strains or species of the genus Mycobacterium. The individuals from which the samples are taken may be patients that also have, are suspected of having, or are at risk for developing one or more secondary or tertiary medical conditions, and in particular, a secondary and/or tertiary infection by one or more nonpathogenic species of bacteria, or one or more pathogenic species of fungal or viral origin, or any combination thereof.

Preferably the population of target nucleic acids contained with the nucleic acid of a sample are suitable for primer-dependent amplification, and particularly so, when the nucleic acids are stored, even when stored at less-than- ideal storage conditions, including, for example, storage under ambient temperature (e.g. from 15°C to about 40°C).

In one embodiment, the invention is directed to systems and methods for simply and efficiently extracting nucleic acids from samples and preferably biological samples. The system comprises: a sterile vessel tapered at one end, optionally with no O ring, wherein the tapered end preferably contains a chemical agent that binds nucleic acid; a sterile lysis buffer that, when added to the biological sample, sterilizes the sample and denatures nucleic acid of the sample; at least one sterile wash buffer that does not disrupt binding of nucleic acid to the agent; and an elution buffer that promotes release of nucleic acid from the agent. Preferably the target nucleic acid is specific for a pathogen such as, for example, a bacterium, a virus, or a parasitic or fungal microorganism. Preferred pathogens include the causative agents of cholera, tuberculosis, influenza, SARS, MERS, HIV, AIDS, malaria or measles. The sample may be a solid, liquid, semi-solid, gel, paste, dispersion, or dense or concentrated gas. Preferably the biological sample is a sample of fluid or tissue from a person suspecting of being infected by or at risk of infection from the pathogen. Preferably the lysis buffer contains a chaotrope, an anionic detergent, a reducing agent, a chelator, a surfactant or antifoaming agent, nuclease-free water, a pH buffer, and no enzymes and the lysis buffer and/or the at least one wash buffer contains a control nucleic, which improves the efficiency of extraction of nucleic acid and/or serves as a positive control of extraction efficiency. Preferably the at least one wash buffer contains a salt and a buffering agent, the elution buffer contains a salt and a buffering agent, and the agent is a silica dioxide wafer. The invention may further comprise a sterile alcohol, such as ethanol, a testing buffer that contains an osmolarity agent, a chelator, magnesium ions, a dye, a mixture of deoxynucleotide triphosphates, a pH buffer, primers pairs specific for PCR amplification of the sequence of the target nucleic acid and optionally a heat-stable polymerase. The invention may further comprise a sterile device for collection of the biological sample and a sterile vessel for transporting the biological sample collected, preferably wherein the sterile vessel contains lysis buffer that sterilizes the biological sample.

The methods of the invention comprise: adding a biological sample containing a target sequence to a sterile vessel containing a lysis buffer forming a sterile mixture; adding the sterile mixture to a sterile cylindrical vessel tapered at one end and with or without an O ring, wherein the tapered end contains an agent that binds to nucleic acid; subjecting the sterile mixture to centrifugation so that the mixture passes through the agent and nucleic acid of the mixture binds to the agent; contacting the agent with at least one wash buffer that does not interfere with binding of nucleic acid to the agent; and contacting the agent with an elution buffer that causes a release of nucleic acid from the agent such that nucleic acid collects in the elution buffer forming an elution mixture. Preferably the biological sample is fluid or tissue from a person suspecting of or at risk of infection by a pathogen, the target sequence is specific to nucleic acid of the pathogen and the pathogen is a bacterium, a virus, or a parasitic or fungal microorganism such as, for example, a pathogen that is the causative agent of one or more of cholera, tuberculosis, influenza, SARS, MERS, HIV, AIDS, malaria or measles. Preferably the lysis buffer contains a chaotrope, an anionic detergent, a reducing agent, a chelator, a surfactant or antifoaming agent, nuclease-free water, a pH buffer, and no enzymes and the lysis buffer and/or the at least one wash buffer contains a control nucleic that improves extraction efficiency of nucleic acid and/or serves as a positive control of extraction efficiency. Preferably the at least one wash buffer contains a salt and a buffering agent, the elution buffer contains a salt and a buffering agent and the agent is a silica dioxide wafer. Preferably, the invention further comprises the option of adding sterile alcohol to the biological sample prior to centrifugation and does not require an incubation step. Preferably the method further comprise adding a testing buffer to the elution mixture, wherein the testing buffer contains an osmolarity agent, a chelator, magnesium ions, a dye, a mixture of deoxynucleotide triphosphates, a pH buffer, primers pairs specific for PCR amplification of the sequence of the target nucleic acid and a heat-stable polymerase and, the target sequence is amplified, quantitated and sequenced by PCR analysis. Preferably the amplified target sequence has a Ct value that is less than the Ct value of the amplified target sequence obtained from conventional PCR of a biological sample and, also preferably, the Ct value is 10% lower than the Ct value of the amplified sequence obtained from conventional PCR of a biological sample. In some embodiments, the methods further include the step of detecting within the obtained population of pathogen- specific nucleic acids the presence of at least a first pathogen- specific nucleic acid segment by contacting the population with a labeled oligonucleotide detection probe, wherein the presence of a labeled hybridization product is indicative of the presence of one or more pathogen- specific nucleic acid segments in the obtained population of nucleic acids.

The composition may further initially include a known quantity of at least a first internal control such as an internal positive (IPC) nucleic acid segment, preferably about 25 to about 500 nucleotides in length, more preferably about 50 to about 250, or more preferably about 90 to about 150 nucleotides in length, wherein the internal positive control nucleic acid segment does not substantially hybridize to genomic nucleic acids of the host from which the sample was obtained, nor to genomic nucleic acids of a pathogen. Such IPCs increase the efficiency of the extraction and also the release of target nucleic acid from the agent. Control nucleic acids include, for example, single-stranded DNA, double-stranded DNA, single-stranded RNA, double- stranded RNA, and combinations thereof including DNA/RNA combinations.

The systems and methods may also preferably further include at the components necessary for and the steps of: (a) performing at least one thermal 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 obtained population of polynucleotides with a composition that comprises at least a pair of distinct, independently-selected, specific amplification primers, a thermostable polymerase, a first osmolarity agent comprising betaine or another cationic functionalized zwitterionic compound, at least a first reference dye, and a plurality of deoxynucleoside triphosphates to produce at least a first pathogen-specific amplification product; and (b) detecting the presence of the amplification product so produced by contacting it with a first labeled pathogen-specific oligonucleotide detection probe, wherein the presence of a labeled hybridization product is indicative of the presence of one or more pathogen-specific nucleic acid segments in the obtained population of nucleic acids. In such embodiments, the pair of distinct, independently-selected, pathogen- specific amplification primers may preferably include a first oligonucleotide primer of 18 to about 30 nucleotides in length, and a second oligonucleotide primer of 18 to about 30 nucleotides in length, wherein each of the first and second primers specifically hybridize to a first, and a second distinct sequence region, respectively.

In related embodiments, the method of the present invention may further optionally include the step of performing a primer-dependent amplification of at least a first sequence region of the internal positive control nucleic acid segment in the obtained population of polynucleotides, and quantitating the amount of the internal positive control nucleic acid segment present in the obtained population of polynucleotides.

Likewise, the method may further optionally include the step of comparing the amount of the internal positive control nucleic acid segment present in the composition at one or more steps along the analytical process, to the amount of IPC that was present in the original composition before the sample was initially added to the lysis/storage/transport medium, or to the amount of target nucleic acids that were present in the original composition. Such comparison may serve to demonstrate that the amount of IPC still contained in the sample in a downstream point of assay is comparable to, or substantially the same as, the known amount of IPC that was present in the MTM composition before the sample was added to it, and may serve to quantitate the amount of target nucleic acids of interest in the collected samples, or downstream assayed components. Such information may also be indicative of the amount of the nucleic acids remaining in the sample as compared to what was originally present, and may provide an estimate of the degree of sample degradation of the polynucleotides originally present over time.

The amplification product of the internal positive control nucleic acid segment may be detected with a suitable oligonucleotide detection probe comprising a first detectable label, and the amplification product of the pathogen-specific nucleic acid segment is detected with an oligonucleotide detection probe comprising a second distinct detectable label. Such method may also further optionally include detecting the presence of one or more drug resistance genes within the population of obtained polynucleotides.

The invention also provides a primer-dependent amplification reaction-compatible composition that preferably includes (a) one or more buffers; (b) one or more osmolality agents; (c) one or more albumin proteins; (d) one or more chelators; (e) one or more salts; (f) at least a pair of distinct, independently-selected, pathogen-specific amplification primers, wherein each of the first and second primers specifically hybridize to a first, and a second distinct sequence region; (g) a pathogen-specific oligonucleotide detection probe comprising a first detectable label, that specifically hybridizes to a third sequence region; (h) at least one primer-dependent amplification reaction-capable thermostable polymerase; and (i) a plurality of deoxynucleoside triphosphates, each present in an amount sufficient to enable the amplification of at least a first pathogen-specific amplification product. Compositions that are thermal-cycling ready (e.g., PCR ready) may be maintained in ready-to-use concentrations, or in concentrated forms such as, for example, 2x, 5x, lOx, 20x 25x, 30x, or greater as convenient or necessary for the particular application. In illustrative embodiments, (a) the one or more buffers preferably include tris(hydroxymethyl)aminomethane (TRIS); (b) the one or more polymerase chain reaction osmolarity agents preferably include Ν,Ν,Ν-trimethylglycine (betaine), dimethyl sulfoxide (DMSO), foramide, glycerol, nonionic detergents, bovine serum albumin (BSA), polyethylene glycol, tetramethylammonium chloride, or any combination thereof; (c) one or more albumin proteins preferably BSA, HAS or any mammalian albumin; (d) the one or more chelators preferably include ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine pentaacetic acid, N,N-bis(carboxymethyl)glycine, ethylenediaminetetraacetic, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof; and (e) the one or more salts preferably include potassium chloride, magnesium sulfate, potassium glutamate, or any combination thereof, and the pair of primers preferably includes: (i) a first oligonucleotide primer of 18 to about 30 nucleotides in length that preferably includes at least a first sequence region that consists of a sequence that is at least 95% identical to the pathogen- specific nucleic acid sequence; and (ii) a second oligonucleotide primer of 18 to about 30 nucleotides in length that preferably includes at least a first sequence region that consists of a sequence that is at least about 90% identical, preferably at least about 95% identical to, and more preferably, at least about 98% identical the pathogen- specific nucleic acid sequence, or a complement thereof.

The pathogen-specific oligonucleotide detection probe preferably is from 24 to about 35 nucleotides in length, and more preferably includes at least a first sequence region that consists of a sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% or greater identical to at least a first contiguous nucleic acid sequence from a pathogen-specific sequence, or a complement thereof. The composition may further optionally include one or more internal reference dyes compatible with a polymerase chain reaction, such as those that include one or more fluorophores, one or more quenchers, one or more reporter molecules, one or more nucleic acid intercalating agents, or any combination thereof.

In illustrative embodiments, the composition at ready-to-use concentrations preferably includes (a) about 50 mM of TRIS; (b) about 70 mM of potassium chloride; (c) about 3 mM of magnesium sulfate; (d) about 45 mM betaine; (e) about 0.03 μg/mL of bovine serum albumin; (f) about 0.1 mM of EDTA; (g) about 0.01 μΜ to about 1 μΜ of dye; (h) about 4 μΜ of a first oligonucleotide primer of 18 to about 30 nucleotides in length; (i) about 4 μΜ of a second oligonucleotide primer of 18 to about 30 nucleotides in length; (j) about 6 μΜ of a pathogen- specific oligonucleotide detection probe of 24 to about 35 nucleotides in length; (k) about 1 unit of Tag polymerase; and (1) about 0.2 mM of deoxynucleoside triphosphates.

The detectable label may preferably include one or more radioactive labels, one or more luminescent labels, one or more chemiluminescent labels, one or more fluorescent labels, one or more phosphorescent labels, one or more magnetic labels, one or more spin-resonance labels, one or more enzymatic labels, or any combination thereof. Exemplary detectable labels include, without limitation, fluorescein, 6-carboxyfluorescein (6-FAM),

6-carboxyfluorescein-N-succinimidyl ester (6-FAMSE), a VIC dye, or any combination thereof.

As noted herein, the invention also provides systems and kits that preferably include one or more of the compositions disclosed herein, and instructions for use in the detection of a pathogen-specific nucleic acid segment in an aqueous sample; optionally the kit may further include (typically in a separate, distinct container), a first MTM composition that comprises: a) one or more chaotropes; b) one or more detergents; c) one or more reducing agents; d) one or more chelators; and e) one or more surfactants, each present in an amount to substantially kill or lyse one or more pathogenic or infected cells, or to denature or inactivate one or more proteins, enzymes, or nucleases liberated there from when placed in the composition for an effective amount of time. In certain embodiments, the kit may also further include (preferably within the MTM composition) a known quantity of at least a first internal positive control nucleic acid segment (and preferably one of from about 50 to about 500 nucleotides in length), wherein the internal positive control nucleic acid segment does not substantially hybridize (and preferably, does not specifically hybridize) to the genomic nucleic acids of the host from which the sample was obtained, nor to genomic nucleic acids of the one or more microbiological pathogens suspected within the sample. As noted herein, such kits may also further optionally include one or more extraction apparatuses for isolating and purifying the population of polynucleotides from the lysed/liberated/denatured sample contacted with the MTM formulation. Such an extraction apparatus may be a portable, bench-top, or even a handheld device that preferably includes: (i) a filtration vessel that has at least one receiving end and that comprises a membrane filter adapted to bind the population of polynucleotides thereto, wherein the membrane filter is disposed at least substantially across a width of the filtration vessel and at least partially therein; and (ii) a volume-dispensing mechanism adapted to controllably dispense and forcibly inject an amount of liquid operably associated with the filtration vessel to filter the liquid there through; and b) instructions for using the extraction apparatus to obtain the population of purified polynucleotides from an aqueous sample suspected of comprising at least a first pathogen. The present invention advantageously improves conventional specimen collection, nucleic acid extraction, ensures complete lysis and sterilization of microbial pathogens contained therein, and facilitates safe and effective transport and storage of such samples from the point of collection to the point of identification and assay. Moreover, the molecular transport media compositions disclosed herein facilitate stabilization of nucleic acids liberated from the collected microorganisms and are also used in the efficient extraction processes of the invention.

Accordingly, the present invention advantageously provides one or more of a collection and preservation formulation that lyses biological pathogens, stabilizes the liberated nucleic acids (both RNAs and DNAs), provides highly efficient and complete extraction of nucleic acids from biological samples, maintains the integrity of the collected nucleic acids such that at least a first portion of which is readily available, and provides nucleic acids suited for downstream molecular diagnostic analysis.

The "one-step" isolation/extraction/storage/transport systems, kits and formulations disclosed herein advantageously accomplish at least one or more, and preferably, all of, the following principal functions: inactivation or killing of pathogens within the sample; lysis of cells and release of nucleic acids from within the cells; inactivation of cellular enzymes, including endogenous and exogenous nucleases, to prevent degradation of the liberated nucleic acids; facilitation of facile collection and safe handling/transport of the sample of isolated polynucleotides at ambient temperatures for extended periods of time without the need for refrigeration or conventional refrigeration or sub-zero storage temperatures; effective stabilization of the nucleic acids during subsequent handling, transport and/or storage of the sample; and preservation and/or maintenance of the integrity of at least a first portion of the population of polynucleotides contained therein for a time sufficient to permit molecular characterization and identification of at least a first nucleic acid segment contained therein.

Preferably, the sample collection, extraction, transport and/or storage systems, kits and methods can be maintained for extended periods of time (from a few hours to a few days, or even a few weeks or months or more) at ambient environmental temperatures, such that the samples and nucleic acids extracted do not require refrigeration for subsequent molecular testing.

Samples to be analyzed may be obtained from a biological samples, with biological samples obtained from a mammal (including e.g., humans, non-human primates, domesticated livestock, and the like). The time between sample collection, nucleic acid extraction, isolation of nucleic acids and amplification/detection analysis of the target nucleic acid sequence of interest may be extended such as, for example, days, weeks and months with no or substantially no degradation and/or degeneration. In some embodiments, the population of nucleic acids obtained from the biological sample is further analyzed. 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 ambient 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 ambient 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 "PrimeMix®," and in some instances "PrimeMix® Universal MTB," can also be used to identify strains of a viral or bacterial sequence, or even species-specific tuberculin strains.

A further embodiment can include a composition including at least one microbial-specific nucleic acid sequence or a biological sample suspected of containing at least one microbial- specific nucleic acid sequence; a solution comprising: (i) one or more buffers (each preferably present in the composition in an amount from about 1 mM to about 1M); (ii) one or more osmolarity agents or albumin proteins at least one of which comprises betaine (each preferably present in the composition in an amount from about 1 mM to about 1M); (iii) one or more chelators (each preferably present in the composition in an amount from about 0.01 mM to about 1 mM); (iv) one or more reference dyes (each preferably present in the composition in an amount from about 0.01 μΜ to about 50 mM, more preferably about 0.02 μΜ to about 1 μΜ); and (v) one or more salts (each preferably present in the composition in an amount from about 50 mM to about 1 M); and a first pair of pathogen-specific amplification primers. In some embodiments, the composition further includes a pathogen-specific probe. In one embodiment, the reference dye is present in an amount of about 0.01 μΜ to about 1 μΜ. Preferably the composition includes one or more salts. The salts are preferably potassium chloride, magnesium chloride, magnesium sulfate, potassium glutamate, or any combination thereof. Preferably, the concentration of salt in the composition is between about 0.5 mM and about 50 mM.

The inclusion of one or more buffers is desirable to control the pH of the formulations which stabilizes the nucleic acids and the enzymes. A preferred pH range is from about 6.0 to about 9.5, preferably between about 6.5 and about 8.0, and more preferably between bout 6.5 and about 7.5. Preferably, the pH of the buffer and/or the overall composition is within one unit of the pKa of the buffer, more preferably within about 0.5 units, more preferably within about 0.2 units and more preferably within about 0.1 units, all as measured at a selected temperature, preferably an ambient temperature. Exemplary buffers include, without limitation, tris(hydroxymethyl) aminomethane (Tris), citrate, 2-(N-morpholino)ethanesulfonic acid (MES), N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), l,3-bis(tris(hydroxymethyl) methylamino)propane (Bis-Tris), 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), N,N-bis(2-hydroxyethyl) glycine (Bicine), N-[tris(hydroxymethyl)methyl]glycine (Tricine), N-2-acetamido-2-iminodiacetic acid (ADA), N- (2-acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES), bicarbonate, phosphate, or any combination thereof. In a preferred embodiment, the buffer includes TRIS.

At least a first osmolality agent can be used within the method to optimize reaction conditions, especially when a high content of guanine and cytosine are present in the sequences, and can include, without limitation, betaine, trimethylglycine, glycine betaine, dimethylsulfoxide (DMSO), foramide, deoxyinosine, glycerine, 7-deaza deoxyguanosine triphosphate, or sodium hydroxide, or any combination thereof.

Exemplary chelators include, without limitation, ethylene glycol tetraacetic acid (EOT A), hydroxy ethylethylenediaminetriacetic acid (HEDTA), diethylene triamine pentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (NTA), ethylenediaminetetraacetic (EDTA), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, potassium citrate, magnesium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof. In preferred embodiments, the chelator includes EDTA, a citrate, or a combination thereof. In a more preferred embodiment, the chelator includes EDTA.

At least a first reference dye, preferably an inert chemical, can optionally be used within the method to normalize the results obtained when using fluorescent compounds, such as those used in FRET technologies. The reference dye, when included, can provide an internal reference to which the reporter dye signal can be normalized. Such a reference dye can include, without limitation, passive reference dyes such as fluorescein, 5-carboxy-X-rhodamine and commercial formulations such as ROX™, or a combination thereof. In a more preferred embodiment, the reference dye includes ROX™.

Preferably, the compositions further include the addition of deoxynucleotide triphosphates (dNTPs), such as deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, deoxythymidine triphosphate, or deoxyurosine triphosphate, or a combination thereof, in an amount from about 0.1 mM to about 50 mM. The compositions of the invention can further include one or more additional compounds or reagents including, but not limited to, albumin. Albumin refers generally to any protein that is water soluble, is moderately soluble in concentrated salt solutions, and experiences heat denaturation. Albumins are commonly found in blood plasma and are unique from other blood proteins in that they are not glycosylated. Preferably the albumin is bovine serum albumin (BSA), magnesium sulfate, water and acids or bases, such as hydrochloric acid and sodium hydroxide. The acids or bases can be added to the final solution to adjust the pH. Preferably, BSA is added in a concentration of about 0.01 μg/μL to about 0.5 μg/μL.

The compositions of the invention can further include one or more polymerases. The one or more polymerases can include, but are not limited to, Taq polymerase, and high fidelity polymerases. Preferably, the one or more polymerases are present in an amount of about 1 U of enzyme to about 10 through about 50 μL· of final solution.

In particular embodiments, the composition will further preferably include at least a first oligonucleotide detection probe that includes a radioactive, luminescent, chemiluminescent, fluorescent, enzymatic, magnetic, or spin-resonance label, or combination thereof. Fluorescent labels can include fluorescein, 6-carboxyfluorescein (6-FAM), or 6-carboxyfluorescein-N- succinimidyl ester (6-FAMSE), or the like, or a combination thereof. Preferred primer and/or probe concentration for each nucleic acid is between about 1 pmol and about 10 μΜ.

The invention further provides for a method for detecting the presence or absence of a pathogen-specific nucleic acid segment in a population of polynucleotides obtained from a biological sample, the method including: (a) performing at least one thermal 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 a population of polynucleotides obtained from a biological sample suspected of containing a pathogen-specific nucleic acid segment with a composition that comprises at least a pair of distinct, independently-selected, pathogen-specific amplification primers, a polymerase, a first osmolality agent comprising betaine, optionally (but preferably) at least a first reference dye, and a plurality of deoxynucleoside triphosphates to produce a pathogen-specific amplification product when a pathogen-specific nucleic acid segment is present in the sample; and (b) detecting the presence of the amplification product by contacting the amplification product with a pathogen-specific oligonucleotide detection probe comprising a first detectable label, wherein the presence of a labeled hybridization product is indicative of the presence of one or more pathogen-specific nucleic acid segments in the population of polynucleotides, wherein the pair of distinct, independently-selected, pathogen-specific amplification primers comprises a first oligonucleotide primer of 18 to about 30 nucleotides in length, and a second oligonucleotide primer of 18 to about 30 nucleotides in length, wherein each of the first and second primers specifically hybridize to a first, and a second distinct sequence region, respectively, within the pathogen-specific sequence, or the complement or reverse complement thereof.

Exemplary formulations of the Mycobacterium PrimeMix® of the invention are described in the examples herein, and include, without limitation, a composition that includes: (a) about 1 U of Taq Polymerase; (b) about 6 μΜ of the detection probe; (c) about 4 μΜ of a reverse oligonucleotide primer of less than about 50, preferably less than about 40, and more preferably still, less than about 30 nucleotides in length; (d) about 4 μΜ of a forward oligonucleotide primer of less than about 50, preferably less than about 40, and more preferably still, less than about 30 nucleotides in length; (e) about 50 mM of Tris; (f) about 70 mM of KC1; (g) about 3 mM of MgS04; (h) about 45 mM of Betaine; (i) about 0.05 μΜ of ROX or comparable reference dye; (j) about 0.025 μg/μl of ultra pure BSA; (k) about 0.2 mM of dNTPs; and (1) about 0.1 mM of EDTA.

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, efficiently extracting nucleic acid, 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 a further embodiment, the invention provides a method for detecting the presence or absence of, for example, a Mycobacterial-speciiic nucleic acid segment, and in particular aspects, provides a method for detecting the presence or absence of a particular type, subtype, or strain of M. tuberculosis. In exemplary embodiments, the invention provides a method of identifying Mycobacterial species and strains that contain one or more IS6110-specific nucleic acid segments in a population of polynucleotides that is preferably obtained from a biological sample.

In another aspect, the present invention provides a method for rapidly detecting in a biological sample, a particular polynucleotide sequence, such as that of the Mycobacterium- specific IS6110 sequence. In an overall and general sense, this method comprises 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.

The label on the probe can include, without limitation, radioactive, luminescent, chemiluminescent, fluorescent, enzymatic, magnetic, or spin-resonance labels known to those of ordinary skill in the molecular arts. In illustrative embodiments, the labeled probe contains at least a first minor groove binder. 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 comprising 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 comprise 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 of ordinary skill 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.

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" (IPCs) into the population of polynucleotides to be isolated, to further ensure the integrity, fidelity, and/or accuracy of the disclosed method.

In certain embodiments, the addition of nucleic acids (e.g., RNA and/or DNA) is contemplated to be beneficial for a variety of purposes and applications of the disclosed methods: a) as a "carrier" (The addition of small amounts of supplemental RNA/DNA has been previously been shown to augment/increase the overall yield of samples/specimens, particularly original specimens that may contain low amounts of target, i.e., cells, viruses, bacteria); b) as an IPC for downstream molecular processes and to track or monitor the fidelity of the nucleic acid preparation from sample collection to detection; and c) for comparison to a 'calibrator' for downstream quantitative analysis, e.g., qRT-PCR and the like. In such embodiments, one or more known or "control" nucleic acids could be added to the compositions in a final concentration of from about 1 ag to about 1 mg, more preferably from about 1 fg to about 1 μg, and more preferably still, from about 1 pg to about 1 ng.

In an illustrative embodiment, the invention provides an isolated single-stranded (ss) or double- stranded (ds) RNA, DNA, PNA, or hybrid thereof that is useful: (a) as a carrier molecule for aiding in the recovery of polynucleotides from a biological sample suspected of containing nucleic acids, and/or (b) as an IPC (i.e., a "known," "reporter," "control," "standard," or "marker") sequence to monitor the integrity and fidelity of specimen collection and polynucleotide isolation/stabilization. In certain embodiments, the invention provides an isolated ds-RNA, ds-DNA, ds-PNA, or a hybrid thereof that is useful as a carrier molecule and/or an IPC. In other embodiments, the invention provides an isolated ssRNA, ssDNA, ssPNA, or a hybrid thereof that is useful as a carrier molecule and/or as an IPC sequence. In exemplary embodiments, the invention provides an isolated ssRNA molecule that is useful as both a carrier molecule and an IPC sequence.

Such molecules can be isolated from natural sources, prepared in the laboratory, or alternatively, a hybrid containing both native- and non-native sequences. As noted herein, because the compositions of the invention are particularly useful for the isolation and characterization of biological specimens obtained from mammalian (and in particular, human) sources that are suspected of containing polynucleotides of pathogen-origin, it is preferable that the sequence(s) employed as carrier and/or positive control compounds substantially contain a primary nucleotide sequence that is not ordinarily found within the genome of a mammal, or within the genome of an organism that is pathogenic to such a mammal. Exemplary mammals include, without limitation, bovines, ovines, porcines, lupines, canines, equines, felines, ursines, murines, leonines, leporines, hircines, and non-human primates.

Preferably, this non-mammalian, non-pathogen-specific carrier/reporter sequence is not cross-reactive, i.e., does not substantially, or preferably, does not, hybridize to, mammalian or pathogen-specific sequences, and as such, non-coding, non-degenerate (i.e., nonsense) sequences are particularly preferred in the formulation of control/carrier sequences to minimize hybridization of the control/carrier sequence to a member of the isolated population of polynucleotides obtained from the collected specimen. Exemplary carrier/control sequences therefore, do not substantially, or preferably, does not, bind (e.g., hybridize under stringent hybridization conditions) to a population of polynucleotides isolated from a mammalian genome, or to a population of polynucleotides isolated from the genome of a bacterium, fungus, virus that is pathogenic to a mammal. Exemplary stringent hybridization conditions known to those of ordinary skill in the art include, without limitation, (a) pre-washing in a solution containing about 5X SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0); (b) hybridizing at a temperature of from about 60°C to about 70°C in 5X SSC overnight; and (c) subsequently washing at about 65 to about 70°C for 20 min. with each of 2X, 0.5X and 0.2X SSC containing 0.1 % SDS), or equivalent hybridization conditions thereto.

Another aspect of the invention provides for a reagent mixture incorporating the aforementioned primers and probes, and kits comprising such compositions for performance of a thermal cycling amplification method. In one embodiment, the invention provides a diagnostic nucleic acid amplification/detection kit that generally includes, in a suitable container, a pathogen-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, a pathogen-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.

Another embodiment of the invention is directed to an apparatus comprising: a sterile vessel cylindrical in shape and configured for addition of a biological sample at one open end and tapered at another open end, wherein the tapered end contains an agent that binds a macromolecule. Preferably the vessel contains no ring, such as an O ring, is preferably cylindrical in shape and configured for centrifugation such that upon centrifugation the biological sample would move towards the agent. Preferably the inner walls of the vessel toward the tapered end are smooth with no ridges that would trap unwanted lysis and/or wash buffers containing contaminating materials. Also preferably, the agent is a silica dioxide wafer or other form that is larger than the another open end. The vessel optionally may contain a cap at either or both ends that is removable or otherwise open-able and/or close-able. Preferably the apparatus is a spin-column for extracting nucleic acid from a biological sample.

Another embodiment of the invention provides a kit for the collection and/or storage, and/or transport of the biological sample prior to genetic analysis of nucleic acids encompassed therein. The present invention allows for a minimal collection of biological material such as sputum, i.e., about 0.01 mL to about 25 mL may be used, preferably about 0.05 mL to about 10 mL, more preferably 0.1 mL to about 5 mL. In such embodiments, a kit preferably includes 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, such as those described above.

In further embodiments, the kits of the invention may also optionally further include one or more extraction devices or apparatuses, as described above, to facilitate the isolation or separation of the nucleic acids from the collected biological sample. 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.

The diagnostic reagents and kits of the present invention may be packaged for commercial distribution, and may further optionally include one or more collection, delivery, transportation, extraction, and/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 aliquotted 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.

The compositions of the invention may be formulated such that the entire specimen collection and nucleic acid extraction, amplification and/or 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 a mixture with all of the necessary isolation, storage, and polynucleotide stabilization components, as well as mixtures with 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 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 commercially available. 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.

The compositions and methods of the present invention are directed to the collection of a clinical or veterinary specimen or a forensic or environmental sample collection system and may include one or more collection tools and one or more reagents for efficiently: 1) obtaining a high yield of suitable specimen beyond what is currently available in the art; 2) inactivating potentially infectious biological pathogens, such as members of the M. tuberculosis complex, so that they are no longer viable and can be handled; shipped, or transported with minimal fear of pathogen release or contamination; or 3) effectively stabilizing and preserving lysed 'naked' RNA/DNA polymers from hydrolysis or nuclease degradation for prolonged periods at ambient temperatures until samples can be processed at a diagnostic laboratory, and preferably for achieving two or more, or all three, of these goals. The collection solutions of the present invention provide the following benefits: inactivation, killing, and/or lysis of microbes, viruses, or pathogens; destruction and/or inactivation of exogenous or endogenous nucleases, including, without limitation, RNase and/or DNase; compatibility with a variety of conventional nucleic acid extraction, purification, and amplification systems; preservation of RNA and/or DNA integrity within the sample; facilitation of transport and shipping at ambient or tropical temperatures, even over extended periods of time, or extreme temperature variations; and suitability for short- (several hours to several days), intermediate- (several days to several weeks), or long- (several weeks to several months) term storage of the isolated nucleic acids. Suitable compositions (also referred to as "PrimeStore®") and methods can be found in commonly owned U.S. Patent Pub. No. 2009-0312285, filed October 1, 2008 (the entire contents of which is specifically incorporated herein in its entirety by express reference thereto).

While the presence of, integrity of, or sequence fidelity of, a particular nucleic acid sequence obtained from, or utilized in the practice of the present invention may be determined using any conventional methodology known to those of ordinary skill in the molecular arts, in one embodiment, PCR amplification is utilized. Likewise, determination of the integrity of a nucleic acid of interest may include determination of the PCR cycle threshold (CT) under given conditions, and determination of the sequence fidelity, qualitative integrity of collected nucleic acids may be determined by conventional DNA or RNA sequencing methods, including, without limitation, the chemical-based methods of Maxam- Gilbert, the dideoxy chain termination method of Sanger et ah, the dye fluorophore-based method of Mathies et ah, or pyrosequencing techniques as described by Nyren and Ronaghi. For example, nucleotide sequencing may be conducted by cloning purified amplicons using a TOPO® 2.0 Cloning Kit (Invitrogen™) and then sequenced using the BigDye® Terminator v3.1 reagent kit. Unincorporated fluorescent nucleotides can be removed using a DyeEx® 96- well plate kit per manufacturer's recommendations (Qiagen®). Nucleotide sequencing could further be performed using an ABI 3100 Genetic Analyzer (ABI Inc., Foster City, CA, USA).

In some embodiments, the collection solution and methods may further include at least one internal positive control (IPC) to monitor fidelity of the processed samples, to monitor the integrity and fidelity of specimen collection and polynucleotide isolation/stabilization and/or to monitor downstream molecular processes or analysis. Methods include placing at least one IPC nucleic acid segment into the collection solutions of the present invention or combining the IPC nucleic acid segment with the extracted population of polynucleotides to monitor downstream molecular processing of the sample and/or extracted nucleic acid. In some embodiments, the IPC is present as a component of the PrimeStore® solution and, as such is substantially stable, and substantially non-degraded when stored in the solution for extended time periods at ambient temperatures. In these instances, the IPC may be considered part of the population of polynucleotides when extracted from the collection solution.

Preferably, the IPC sequence is not cross-reactive, i.e., does not substantially, or preferably, do(es) not, hybridize to, mammalian or pathogen-specific sequences, and as such, non-coding, non-degenerate {i.e., nonsense) sequences are particularly preferred in the formulation of control/ carrier sequences to minimize hybridization of the control/carrier sequence to a member of the isolated population of polynucleotides obtained from the collected specimen. Exemplary carrier/control sequences therefore, do not substantially, or preferably, do(es) not, bind (e.g., hybridize under stringent hybridization conditions) to a population of polynucleotides isolated from a mammalian genome, or to a population of polynucleotides isolated from the genome of a bacterium, fungus, protozoan, virus that is pathogenic to a mammal.

In certain embodiments, the invention provides an isolated single stranded (ss)-RNA, ss- DNA, ss-PNA, double stranded (ds)-RNA, ds-DNA, ds-PNA, or a hybrid thereof, that is useful as an IPC. In preferred embodiments, where the isolation and detection of M. tuberculosis- complex specific nucleic acid is desired, a single stranded deoxyribonucleic acid segment is used. In illustrative embodiments, the invention provides for IPC sequences that comprise, consist essentially of, or consists of, nucleic acid sequences that are preferably at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or more identical to any sequences of the target pathogen.

Where further molecular processing of the sample or extracted nucleic acid comprises extraction and/or identification of M. tuberculosis-complex specific nucleic acids, the IPC sequences of the present invention should contain at least a first sequence domain that specifically hybridizes (i.e., binds) to a suitably-detectable probe, including, without limitation, molecularly-labeled probes and derivatives thereof. Exemplary labeled probes are those that include radioactive, luminescent, chemiluminescent, fluorescent, enzymatic, magnetic, or spin- resonance labels known to those of ordinary skill in the molecular arts. In preferred embodiments, the probe is labeled with 6-FAM or VIC™ dye. In illustrative embodiments, the labeled probe contains at least a first minor groove binder. In further embodiments, wherein amplification strategies such as PCR will be employed, the IPC sequences of the present invention contain at least a second sequence domain that specifically binds to a forward PCR amplification primer and a third sequence domain that specifically binds to a reverse PCR amplification primer.

Following collection of nucleic acids from a biological sample, extraction from the collection solution and microorganism debris, such as proteins, lipids and carbohydrates, may be performed by systems and methods of the invention. Conventional methods used include commercially available nucleic acid extraction compositions and methods, such as, but not limited to QiaAmp® DNA Mini kit (Qiagen®, Hilden, Germany), MagNA Pure 96 System (Roche Diagnostics, USA), and the NucliSENS® easyMAG® extraction system (bioMerieux, France). Systems and methods of the present invention include extraction of genomic nucleic acid 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.

In exemplary compositions and methods of the invention, primers and probes of the invention are added to a particular formulation so that PCR may be performed. Preferably, about 8 μΜ of forward and reverse primers, about 6 μΜ of probe and about 1 unit of Taq are present in PrimeMix®. Exemplary concentration ranges of additional components of PrimeMix® can be seen in Table 1A and PrimeStore® in Table IB.

TABLE 1 A

FORMULATION RANGES OF EXEMPLARY COMPONENTS FOR THE PREPARATION

OF PRIMEMIX® COMPOSITIONS

Reagent Component Final Concentration Ranges

1. One or more buffers, e.g. : about 1 mM to about 1 M

Tris, citrate, MES, BES, Bis-Tris,

HEPES, MOPS, Bicine, Tricine, ADA,

ACES, PIPES, bicarbonate, phosphate

2. One or more polymerase chain reaction

osmolarity agents, cationic functionalized zwitterionic compounds, <^?.g. :about ImM to about 1 M betaine, DMSO, foramide, glycerol,

nonionic detergents, BSA, polyethylene

glycol, tetramethylammonium chloride

3. One or more chelators, e.g. : about 0.01 mM to about 1 mM

EGTA, HEDTA, DTPA, NTA, EDTA,

citrate anhydrous, sodium citrate, calcium

citrate, ammonium citrate, ammonium bicitrate,

citric acid, diammonium citrate, potassium

citrate, magnesium citrate, ferric ammonium

citrate, lithium citrate

4. One or more dyes, e.g. : about 0.01 mM to about 50 mM

fluorescein, 5-carboxy-X-rhodamine, ROX™

5. One or more salts, e.g. : about 50 mM to about 1 M

potassium chloride, magnesium sulfate,

potassium glutamate

6. One or more polymerases, e.g. : about 0.05U to about 1U

Taq, Pfu, KOD,

Hot start polymerases, next gen. polymerases

7. Deoxynucleoside triphosphates, e.g. : about 0.1 mM to about 1 mM

dATP, dTTP, dGTP, dCTP, dUTP

Preferably, to this formulation a sufficient amount of primers and probe are added so as to amplify and detect the desired target.

2-amino-2-hydroxymethyl-propane-l,3-diol (TRIS) was obtained from Applied

Biosystems/Ambion (Austin, TX, USA). 2-[2-(Bis(carboxymethyl)amino)ethyl-

(carboxymethyl)amino]acetic acid (EDTA) GIBCO® UltraPure BSA was obtained from Invitrogen™ Corp. (Carlsbad, CA, USA). All other reagents are available commercially from Sigma- Aldrich or USB Corporation.

Table 1 B

FORMULATION RANGES OF EXEMPLARY COMPONENTS FOR THE PREPARATION OF PRIMESTORE™ COMPOSITIONS

Reagent Component Final Concentration ranges

1. A chaotrope, e.g.:

Guanidine thiocyanate about 0.5 M to about 6 M

or Guanidine hydrochloride about 0.5 M to about 6 M

or Guanidine isocyanate about 0.5 M to about 6 M

2. An anionic detergent, e.g.:

N-lauroyl sarcosine (inter alia Na salt) about 0.15% to about 1% (wt./vol.) or Sodium dodecyl sulfate, about 0.15% to about 1% (wt./vol.)

Lithium dodecyl sulfate, about 0.15% to about 1% (wt./vol.)

Sodium glycocholate, about 0.15% to about 1% (wt./vol.)

Sodium deoxycholate, about 0.15% to about 1% (wt./vol.)

Sodium taurodeoxycholate, or about 0.15% to about 1% (wt./vol.)

Sodium cholate about 0.1% to about 1% (wt./vol.)

3. A reducing agent, e.g. :

TCEP about 0.5 mM to about 30 mM

or β-ΜΕ, DTT, formamide, or DMSO about 0.05 M to about 0.3 M

4. A chelator, e.g.:

Sodium citrate about 0.5 mM to about 50 mM

or EDTA, EGTA, HEDTA, DTPA, NTA, or APCA about 0.01 mM to about 1 mM

5. A buffer (e.g., TRIS, HEPES, MOPS, MES, Bis-Tris, etc.) about 1 mM to about 1 M

6. An acid (e.g., HC1 or citric acid) q.s. to adjust to a pH of about 6 to 7,

preferably 6.4 to 6.8

7. Nuclease-free water q.s. to desired final volume

Optionally one or more of:

8. A surfactant/defoaming agent, e.g.:

Antifoam A® or Tween® about 0.0001 % to about 0.3% (wt./vol.)

9. An alkanol (e.g., methanol, ethanol, propanol, etc.) about 1% to about 25% (vol./vol.)

10. RNA or DNA about 1 pg to about 1 μg/mL

Preferably, the PrimeStore composition contains a resin that facilitates the breakdown of the components of the biological sample and release of nucleic acid. For example, components that break down samples and cause the release of nucleic acid include, preferably, agarose, glass, cellulose, polyacrylamide, sepharose, sephadex, silica, or another matrix media. Affinity beads are preferably magnetic beads such as, for example, beads commercially available which may bind to macromolecules such as, for example, nucleic acids (DNA and/or RNA), proteins, lipids, fatty acids, carbohydrates and peptides. Beads may contain pores of defined sizes that are useful for inclusion or exclusion molecular size chromatography. Matrix media such as, for example, resins that are useful with the compositions and method of the invention such as, preferably, amino acid resins, carbohydrate resins, ion exchange resins, and hydrophobic and hydrophilic resins. The presence of matrix material in the composition of the invention serve to facilitate the release and subsequent capture of macromolecules from the cells, cell structures, macromolecules, and biological and non-biological debris of the sample. Preferably, these same matrix materials can then serve to expedite the isolation of the macromolecules for later analysis through magnetic attraction, molecular affinity, ionic or non-ionic interactions, density or specific density, hydrophobic or hydrophilic interactions, shape, color or light emission or absorption or any unique or identifiable distinguishing chemical or physical property. When matrix material is utilized, the sterile vessel may contain a screen or filter such that the matrix material does not reach the nucleic acid binding agent. Alternatively, the matrix material may be the nucleic acid binding agent. Also preferably, the PrimeStore and PrimeMix compositions, and other compositions of the invention, contain no ingredients that inhibit subsequent nucleic acid testing. Substances that inhibit nucleic acid testing include gelatin type and concentration. For example, porcine and bovine gelatins, at concentrations from 1.0 to 0.1%, are inhibitory to silica based extraction, whereas bovine gelatin of varying Bloom values (e.g., 75-225 = low to high Bloom) are relatively neutral to extraction.

Compositions and Methods for Multiplex Analysis of Biological Samples

In some embodiments, it may be desirable to provide reagent mixtures that include more than a single pair of amplification primers and a detection probe that is specific for a given target nucleic acid sequence. For example, when it is desirable to determine the presence of two or more different types of pathogens, the composition of the invention may be formulated to contain a first pair of amplification primers that specifically bind to at least a first target region of one pathogen-specific polynucleotide, and a second pair of amplification primers that specifically bind to at least a first target region of another pathogen- specific polynucleotide.

Alternatively, when it is desirable to determine the presence of two or more different strains, the composition of the invention may be formulated to contain a first pair of amplification primers that specifically bind to at least a first target region of a particular pathogen-specific polynucleotide, and a second pair of amplification primers that specifically bind to at least a first target region of a second, distinct pathogen-specific polynucleotide.

Additionally, when it is desirable to determine the presence of one or more additional microorganisms, i.e., to identify whether a patient is co-infected, with other bacterial, or fungal, or viral infections, for example, gram-positive and gram-negative bacteria, human immunodeficiency virus, pneumoccocus, influenza, Yesinia pestis, Pseudomonas sp., Stenotrophomonas maltophilia, Burkholderia cepacia, Streptococcus sp., Moraxella catarrhalis, Enterobacteriaceae, Haemophilus sp., Staphylococcus sp., Rhinovirus, Respiratory syncytial virus, Coronavirus, Adenovirus, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Pneumocystis jiroveci, and the like.

In some instances, it is desirable to test for drug resistance genes or mutations within the M. tuberculosis complex-specific polynucleotide. Multi-drug resistant (MDR)-TB strains could arise as a consequence of sequential accumulation of mutations conferring resistance to single agents, or by a single step process such as acquisition of an MDR element. A series of distinct mutations conferring resistance to Rifampin, INH, Streptomycin, Ethambutol, ETH, PZA, Kanamycin, and quinolones has been identified. Some of these MDR isolates arise because random mutations in genes that encode targets for the individual anti-microbial agents are selected by sub-therapeutic drug levels resulting from treatment errors, poor adherence to treatment protocols, or other factors.

In these embodiments, the composition of the invention may be formulated to contain a first pair of amplification primers that specifically bind to at least a first target region of a particular pathogen- specific polynucleotide, and a second pair of amplification primers that specifically bind to at least a first target region of a drug resistance-polynucleotide found within, for example, multi-drug resistant strains or extensively-drug resistance strains. For example, this can include resistance to rifampicin and/or isoniazid (resistance to these first-line anti-TB drugs classically defines a multi-drug resistant [MDR] tuberculosis), as well as to one or more members of the quinolone family, or kanamycin, capreomycin or amikacin, or any combination thereof.

For detection of the particular amplification product(s) produced from such compositions, the compositions will also further include a first detection probe that specifically binds to the amplification product produced from the first pair of amplification primers, and a second distinct detection probe that specifically binds to the amplification product produced from the second pair of amplification primers. In such compositions, it is preferable that the two, three or four detection probes present in the formulation be distinct, such that each of the probes (if specifically bound to a target in the resulting amplification mixture) may be individually detectable using conventional methodologies. Such probe distinctiveness is readily achievable in the conventional arts, using, for example, detection probes that include detection moieties that fluoresce at two, three or four distinctly-different wavelengths.

In some aspects of the invention, the amplification and/or detection of target nucleic acids may be done sequentially, while in other aspects, it may be desirable to amplify and/or detection multiple target nucleic acids simultaneously. For example, a given biological sample could first be screened for the presence of M. tuberculosis -specific target sequence(s), and if none are found, the sample then secondarily screened for the presence of M. bovis, M. africanum, M. microti, M. cannetti, M. caprae and M. pinnipedi-speciiic target sequence(s).

The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.

Examples

To improve and simplify extraction of RNA/DNA from specimens, an extraction system was designed that is more rapid, requires fewer steps to complete and utilizes less equipment. Extraction efficiency is equal to or better than currently used RNA/DNA extraction methods.

In these experiments, an equal concentration of Mycobacterium tuberculosis H37Rv culture (10³ CFU/mL) was added to each extraction kit and subsequently extracted and subjected to real-time PCR analysis. The QiaAmp DNA Mini kit was used according to manufacturer's recommendations as outlined per extraction of cultured cells. For DNA/RNA extraction using PrimeXtract, a mixture of 10³ MTB, PrimeXtract lysis buffer, and 100% ethanol were added in equal volumes, vortexed, and subjected to extraction as suggested in the User's Instructions. Elution for both extraction kits was 100 μΕ of nuclease free water (Ambion /LifeTechnologies, Austin, TX). All reactions were performed in triplicate. A total of 2.5 μΕ of extracted material was amplified using PrimeMix MTB Complex IX Amplification Solution (Longhorn Vaccines & Diagnostics, San Antonio, TX) on an ABI 7500 Real Time PCR System (Applied Biosystems/LifeTechnologies, CA) using established thermo cycling and analysis parameters.

As shown in Figure 1, equal concentrations of Mycobacterium tuberculosis DNA was extracted using all of the reagents/buffers supplied in the QiaAmp DNA Mini Kit (The QIAamp DNA Mini Kit provides silica- membrane-based nucleic acid purification from tissues, swabs, CSF, blood, body fluids, or washed cells from urine. No mechanical homogenization is necessary as the tissues are lysed enzymatically. The convenient spin-column procedure reduces hands-on preparation time to 20 minutes. Purification of DNA/RNA using the QIAamp DNA Mini Kit can be automated on the QIAcube; For 50 DNA preps: 50 QIAamp Mini Spin Columns, QIAGEN Proteinase K, Reagents, Buffers, Collection Tubes (2 ml)), i.e., Lysis Buffer AL and AW1 and AW2 wash buffers with either the: 1) Qiagen or 2) PrimeXtract spin columns. In this comparison all reagents were equal except for the spin column used for extraction and binding of nucleic acids. PrimeXtract columns demonstrated improvement in comparison to Qiagen spin columns as evident by reduced real-time PCR cycle threshold values (i.e., 27.3 for PrimeXtract vs. 28.6 for Qiagen; Note: lower CT values are an indication of more initial DNA/RNA template since CT values are directly related to increased DNA/RNA template from extraction).

As shown in Figure 2, Mycobacterium tuberculosis was processed using: 1) Qiagen or 2) PrimeXtact spin columns and subsequently processed and extracted using PrimeXtact Lysis, Wash I, and Wash II buffers. In this example, PrimeXtract columns show equivalent results when compared to the Qiagen columns.

Shown in Figure 3, Mycobacterium tuberculosis was extracted with: 1) Qiagen or 2) PrimeXtact kits using the respective manufacturer' s recommended protocol. The concentration of MTB bacteria that was processed (i.e., 10³ CFU/mL) and the output elution volume was equal for both kits. In this example, PrimeXtract showed increased sensitivity (i.e., lower CT values) compared to the industry standard bench top extraction kit.

Figures 4 and 5 depict the spin columns used in this example. Figure 4 represents the conventional spin column and Figure 5, the spin column of the invention. Spin columns of the invention are designed to ensure complete elution with no lysis/wash buffer carryover. This results in contaminate-free, high-purity RNA and DNA for downstream molecular biology.

There are several advantages of PrimeXtract over the Qiagen kit. The Qiagen kit requires the addition of proteinase K to the sample for the lysis step. PrimeXtract does not require proteinase K to achieve lysis of the cells (one less step for the technician and also less training required). The Qiagen kit also requires a 10 minute incubation step at 56°C (to include lysis buffer with proteinase K) for efficient lysis of the sample. PrimeXtract lysis (using PrimeStore) does not require a heated incubation step. (There is no need for a heat block and the extra 10 minute incubation adds to the length of the extraction process). The Qiagen kit adds proteinase K, lysis buffer, and 100% ethanol in three separate steps. Each step is followed by vortexing and brief centrifugation. All of these individual steps add time to the procedure from opening caps, adding reagent/buffer, closing caps, vortexing, placing in the centrifuge for brief spin and then removing from the centrifuge for the next step in the protocol. The PrimeXtract protocol adds sample, lysis, and ethanol all together with one vortex and brief centrifugation. Additionally, the Qiagen protocol requires a minimum sample input volume of 200μL·. If the sample is less than 200μL· an additional step is involved in bringing the sample up to 200μL· with an appropriate volume of phosphate buffered saline (PBS) before adding the proteinase K and lysis buffer. The PrimeXtract system does not require a minimum sample volume to start the procedure. The Qiagen kit uses larger volumes (500μί) of wash buffers (AW1 and AW2) requiring 4 collection tube changes to discard the filtrates. PrimeXtract optionally utilizes only 200μL· of each wash buffer, which involve only 2 collection tube change to discard filtrates. The Qiagen protocol uses 200μL· of elution volume with the kit AE solution or nuclease free water. This volume is necessary to wet the entire surface of the filter in their spin column. PrimeXtract spin column filters are much smaller in diameter and require as little as 20μί of elution volume (1/10* the volume of the Qiagen spin

PrimeXtract DNA/RNA extraction is comparable and marginally superior to the standard Qiagen DNA Mini Kit for extraction of DNA/RNA from cultured cells or primary samples. Since there are no incubations and fewer steps in the extraction, the procedure can be performed in about half the time of the Qiagen kit. Additionally, the method does not require proteinase K (an enzyme), or a heated incubation step reducing the need for additional instrumentation. Since PrimeStore MTM is compatible with PrimeXtract the sample is 'pre-lysed', does not require heated incubation or addition of proteinase K, and can be added directly to the PrimXtract column. Furthermore, the refined extraction spin column is tapered and the does not contain an o-ring, which greatly reduces buffer carryover into the elution.

Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications including U.S. Patent Application Publication No. 2009/0098527; U.S. Patent No. 8,084,443; U.S. Patent No. 8,080,645; U.S. Patent Application Publication No. 2012/0107799; U.S. Patent No. 8,652,782; and U.S. Patent Application Publication No. 2014/0038174, are specifically and entirely incorporated by reference. The term comprising, where ever used, is intended to include the terms consisting and consisting essentially of. Furthermore, the terms comprising, including, and containing are not intended to be limiting. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the claims.

Claims

1. An extraction system for simply and efficiently extracting nucleic acid from a biological sample comprising:

a sterile vessel tapered at one end with no O ring, wherein the tapered end contains an agent that binds nucleic acid;

a sterile lysis buffer that, when added to the biological sample, sterilizes the sample and denatures the nucleic acid of the sample;

at least one sterile wash buffer that does not disrupt binding of the nucleic acid to the agent; and

a sterile elution buffer that promotes release of the nucleic acid from the agent.

2. The system of claim 1, wherein the nucleic acid contains a target sequence that is indicative of the presence of a pathogen.

3. The system of claim 2, wherein the biological sample is a sample of fluid or tissue from a person suspecting of being infected by or at risk of infection from the pathogen.

4. The system of claim 2, wherein the pathogen is a bacterium, a virus, or a parasitic or fungal microorganism.

5. The system of claim 2, wherein the pathogen is the causative agent of cholera, tuberculosis, influenza, SARS, MERS, HIV, AIDS, malaria or measles.

6. The system of claim 1, wherein the sterile lysis buffer contains a chaotrope, an anionic detergent, a reducing agent, a chelator, a surfactant or antifoaming agent, nuclease-free water, a pH buffer, and no enzymes.

7. The system of claim 1, wherein the sterile lysis buffer or the at least one wash sterile buffer contains a control nucleic.

8. The system of claim 7, wherein the control nucleic acid improves extraction of nucleic acid and/or serves as a positive control of extraction efficiency.

9. The system of claim 1, wherein the at least one sterile wash buffer contains a salt and a buffering agent.

10. The system of claim 1, wherein the sterile elution buffer contains a salt and a buffering agent.

11. The system of claim 1, wherein the agent is a silica dioxide wafer.

12. The system of claim 1, further comprising a sterile alcohol.

13. The system of claim 1, further comprising a sterile testing buffer that contains an osmolarity agent, a chelator, magnesium ions, a dye, a mixture of deoxynucleotide triphosphates, a pH buffer, primers pairs specific for PCR amplification of the sequence of the target nucleic acid and optionally a heat-stable polymerase.

14. The system of claim 1, further comprising a sterile device for collection of the biological sample and a sterile vessel for transporting the biological sample collected.

15. The system of claim 14, wherein the sterile vessel contains the lysis buffer that sterilizes the biological sample.

16. A method comprising:

adding a biological sample containing a target sequence to a sterile vessel containing a sterile lysis buffer forming a sterile mixture;

adding the sterile mixture to a sterile cylindrical vessel tapered at one end and without an O ring, wherein the tapered end contains an agent that binds to nucleic acid;

subjecting the sterile mixture to centrifugation so that the mixture passes through the agent and nucleic acid of the mixture binds to the agent;

washing the agent with at least one sterile wash buffer that does not interfere with binding of nucleic acid to the agent; and

contacting the agent with a sterile elution buffer that causes a release of nucleic acid from the agent such that nucleic acid collects in the sterile elution buffer forming an elution mixture.

17. The method of claim 16, wherein the biological sample is fluid or tissue from a person suspecting of or at risk of infection by a pathogen.

18. The method of claim 17, wherein the pathogen is a bacterium, a virus, or a parasitic or fungal microorganism.

19. The method of claim 17, wherein the target sequence is specific to nucleic acid of the pathogen.

20. The method of claim 17, wherein the pathogen is the causative agent of cholera, tuberculosis, influenza, SARS, MERS, HIV, AIDS, malaria or measles.

21. The method of claim 16, wherein the sterile lysis buffer contains a chaotrope, an anionic detergent, a reducing agent, a chelator, a surfactant or antifoaming agent, nuclease-free water, a pH buffer, and no enzymes.

22. The method of claim 16, wherein the sterile lysis buffer or the at least one sterile wash buffer contains a control nucleic.

23. The method of claim 22, wherein the control nucleic acid improves extraction of nucleic acid and/or serves as a positive control of extraction efficiency.

24. The method of claim 16, wherein the at least one sterile wash buffer contains a salt and a buffering agent.

25. The method of claim 16, wherein the sterile elution buffer contains a salt and a buffering agent.

26. The method of claim 16, wherein the agent is a silica dioxide wafer.

27. The method of claim 16, further comprising adding sterile alcohol to the biological sample prior to centrifugation.

28. The method of claim 16, which does not require an incubation step.

29. The method of claim 16, further comprising adding a sterile testing buffer to at least a portion of the elution mixture forming a testing composition, wherein the sterile testing buffer contains an osmolarity agent, a chelator, magnesium ions, a dye, a mixture of deoxynucleotide triphosphates, a pH buffer, primers pairs specific for PCR amplification of the sequence of the target nucleic acid and a heat-stable polymerase.

30. The method of claim 29, further comprising amplifying a target sequence within the testing composition by PCR to form an amplified target sequence.

31. The method of claim 30, further comprising quantitating the amplified target sequence.

32. The method of claim 30, further comprising determining the sequence of the amplified target sequence.

33. The method of claim 30, wherein the amplified target sequence has a Ct value that is less than the Ct value of the amplified target sequence obtained from conventional PCR of the biological sample.

34. The method of claim 33, wherein the Ct value is 10% lower than the Ct value of the amplified target sequence obtained from conventional PCR of the biological sample.

35. An apparatus comprising:

a sterile vessel cylindrical in shape and configured for addition of a biological sample at one open end and tapered at another open end, wherein the tapered end contains an agent that binds a macromolecule.

36. The apparatus of claim 35, wherein the vessel contains no O ring.

37. The apparatus of claim 35, wherein the vessel is cylindrical and configured for centrifugation such that upon centrifugation the biological sample would move towards the agent.

38. The apparatus of claim 35, wherein the inner walls of the vessel toward the tapered end are smooth with no ridges.

39. The apparatus of claim 35, wherein the agent is silica dioxide wafer and the

macromolecule is a nucleic acid.

40. The apparatus of claim 39, wherein the wafer is larger than the another open end.

41. The apparatus of claim 35, which is a spin-column for extracting nucleic acid from a biological sample.

42. The apparatus of claim 35, wherein the agent is a nucleic acid capture matrix material.

43. The apparatus of claim 42, wherein the nucleic acid capture matrix material comprises magnetic beads.

44. The apparatus of claim 35, wherein the biological sample contains a sterile lysis buffer.

45. The apparatus of claim 44, wherein the sterile lysis buffer does not contain a gelatin.