Direct amplification and detection of viral and bacterial pathogens

Abstract

To provide methods for identifying the presence or absence of target nucleic acids from a microorganism, using direct amplification without a nucleic acid extraction step, but retaining substantially the same specificity and sensitivity of methods assaying the extracted nucleic acids.SOLUTION: A method for identifying the presence or absence of a target nucleic acid from a microorganism in a biological sample from a human, the method comprising: (a) contacting the sample with a DNA polymerase and a buffer under conditions suitable for amplification of the target nucleic acid from the sample without extracting the target nucleic acid from the sample; (b) thermocycling the sample from step (a) such that the target nucleic acid, if present, is amplified; and (c) detecting the amplified target nucleic acid, if present, produced from step (b), wherein nucleic acid in the sample is not extracted from the sample prior to amplification.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Patent Application No. 61 / 505,055, filed July 6, 2011, and U.S. Patent Application No. 61 / 552,405, filed October 27, 2011, the disclosures of each of which are considered part of the disclosure of this application and are incorporated by reference into the disclosure of this application.

[0002] The present invention relates to diagnostic and detection methods for nucleic acids of viral and bacterial pathogens using direct amplification. [Background technology]

[0003] The following discussion of the background of the invention is provided merely to aid the reader in understanding the invention and is not intended to describe the invention or to constitute prior art to the invention.

[0004] Clinical detection of the virus is usually achieved using any one of a variety of methods. For example, viral particles or nucleic acids can be isolated from biological samples (e.g., nasopharyngeal aspirates, throat swabs, blood, feces, etc.). Retrospective diagnosis can be made by serology. Complement fixation tests (CFTs) are the most widely used in this method, but hemagglutination inhibition (HAI) and enzyme immunoassays (EIAs) can be used to make type-specific diagnoses. For a more rapid diagnosis, either antigen detection or RNA detection can be performed. Antigen detection can be performed by IFT or EIA, while RNA detection by reverse transcriptase polymerase chain reaction (RT-PCR) is used to achieve the highest level of sensitivity and specificity. However, the latter is expensive and requires advanced technology.

[0005] Similarly, bacterial detection can be achieved using a variety of methods, including Gram staining, culture, microarray, and polymerase chain reaction (PCR) or real-time PCR.Unlike viruses such as influenza, whose genome is composed of RNA and whose detection requires a transcription step to create target cDNA for use in conventional PCR or real-time PCR, bacterial detection can be achieved using standard PCR protocols.Even if it does not require an additional RT step, PCR or real-time PCR is a time-consuming and expensive diagnostic method, as described below.

[0006] RT-PCR is a laboratory technique used to amplify and quantify target nucleic acids. The procedure follows the general principles of the polymerase chain reaction, but in RT-PCR, the RNA strand is first reverse transcribed into its DNA complement (cDNA) using reverse transcriptase, and the resulting cDNA is then amplified using traditional or real-time PCR. The reverse transcription (RT) step can be carried out either in the same tube as the PCR (one-step PCR) or in a separate tube (two-step PCR), using temperatures between about 40°C and 50°C depending on the properties of the reverse transcriptase used. The two strands are then separated by denaturing the dsDNA at about 95°C, allowing the primers to recombine at the lower temperature and initiate a new amplification reaction. DNA extension from the primers occurs using the thermostable Taq DNA polymerase, usually at about 72°C. Real-time RT-PCR offers a method in which the amplicons can be visualized simultaneously as the amplification proceeds, using fluorescent reporter molecules.

[0007] Given that preparation and processing of viral and bacterial nucleic acids from biological samples for detection, diagnosis, and / or quantification requires highly complex steps, there is a need for methods that require fewer steps, are less technically demanding, and can be performed in less time when a rapid diagnosis is required. Summary of the Invention

[0008] The present invention is based on the discovery of a reagent mixture that allows for the direct amplification of a sample, without the step of nucleic acid extraction.

[0009] In one aspect, the present invention provides a method for confirming the presence or absence of a target nucleic acid derived from a microorganism in a biological sample obtained from a human, the method comprising the steps of: (a) contacting the sample with a DNA polymerase and a buffer under conditions suitable for amplifying a target nucleic acid derived from the sample, without extracting the target nucleic acid from the sample; (b) thermal cycling the sample obtained by step (a) such that the target nucleic acid, if present, is amplified; and (c) detecting the amplified target nucleic acid produced by step (b), if present, wherein the nucleic acid of the sample is not extracted prior to amplification.

[0010] In another aspect, the present invention provides a method for determining the presence or absence of a target nucleic acid derived from a microorganism in a biological sample obtained from a human, the method comprising the steps of: (a) contacting the sample with a DNA polymerase and a buffer under conditions suitable for amplifying a target nucleic acid derived from the sample, without extracting the target nucleic acid from the sample; (b) thermal cycling the sample obtained by step (a) such that the target nucleic acid, if present, is amplified; and (c) detecting the amplified target nucleic acid produced by step (b), if present, wherein the nucleic acid in the sample is not extracted from the sample prior to amplification, and wherein the buffer comprises at least one component selected from the group consisting of KCl, bovine serum albumin and a surfactant.

[0011] In some embodiments, the nucleic acids of the sample are not diluted prior to step (a). In further embodiments, the sample is heated prior to step (b), or in yet other embodiments, the sample is heated prior to step (a). The sample may be heated to a temperature of about 70° C. or greater for about 2 minutes or more prior to step (b).

[0012] In further embodiments, the buffer may include potassium chloride (KCl). In some embodiments, KCl may be present at a concentration of about 5 mM to about 50 mM. The buffer may further include GoTaq™ Flexi buffer, which may be present during step (b) at a concentration of 1X to 5X. The buffer may also include bovine serum albumin. The buffer may include a detergent. In some embodiments, the detergent is a cationic detergent. In further embodiments, the DNA polymerase is Taq polymerase. The target nucleic acid may be DNA, or in some embodiments, the target nucleic acid may be RNA. If the sample is RNA, the sample may further be contacted with a reverse transcriptase. The sample may be contacted with the DNA polymerase and the reverse transcriptase simultaneously.

[0013] In further embodiments, the sample is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, oral fluid, and stool. In some embodiments, the sample is whole blood. In some embodiments, the sample is obtained from the oral cavity. In yet another embodiment, the microorganism may be a virus or may be selected from the group consisting of influenza virus, respiratory syncytial virus, herpes simplex virus, and enterovirus. Also, in some embodiments, the microorganism may be a bacterium, for example, in further embodiments, Clostridium difficile (C. difficile).

[0014] As used herein, the term "RNA" refers to a nucleic acid molecule that contains a ribose sugar as opposed to the deoxyribose sugar found in DNA. As used herein, RNA refers to all species or RNAs, including messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), as well as small RNA species that have regulatory functions. "Small RNA species" has a clear meaning and refers to non-translated RNAs that have housekeeping or regulatory roles in bacteria. "Small RNA species" are not rRNA or tRNA.

[0015] As used herein, the term "target nucleic acid" refers to any nucleic acid molecule or fragment that is useful for diagnosing a particular virus or bacterium, including, for example, a pathogenic virus or bacterium. The target nucleic acid may be a DNA or RNA molecule derived from the target species.

[0016] As used herein, the term "thermocycling" refers to any technique that uses laboratory equipment to amplify nucleic acid segments by primer extension reactions that use pre-programmed cycles of temperature increase and decrease. Examples of thermocycling include, but are not limited to, PCR, real-time PCR, and RT-PCR.

[0017] As used herein, the term "reverse transcriptase polymerase chain reaction" or "RT-PCR" refers to any technique for synthesizing and amplifying a DNA molecule having a sequence that is a copy of an RNA sequence. RT-PCR is useful, for example, for detecting RNA species in quantitative analysis of gene expression, and for producing DNA copies of RNA for use in cloning, construction of cDNA libraries, probe synthesis, and signal amplification in in situ hybridization.

[0018] As used herein, the term "reagent mix" refers to a composition having all the elements needed to perform a reverse transcription polymerase chain reaction or a real-time polymerase chain reaction, including, but not limited to, primers with specificity for a diagnostic target RNA or DNA sequence, respectively, and a polymerase.

[0019] As used herein, "primer" refers to a synthetic or natural oligonucleotide that can act as an initiation point for nucleic acid synthesis or replication along a template strand when placed under conditions that allow polymerase to catalyze the synthesis of complementary strand.In the context of reverse transcription, primer is composed of nucleic acid and initiates the amplification of RNA template.In the context of PCR, primer is composed of nucleic acid and initiates the amplification of DNA template.

[0020] As used herein, the term "DNA polymerase" refers to any enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerase acts to add free nucleotides to the 3' end of the newly formed strand, resulting in the extension of the new strand in the 5'-3' direction.

[0021] As used herein, "lysis" refers to the disruption or alteration of cell walls or virus particles that facilitates access to or release of cellular RNA or DNA. Neither complete breakdown or disruption of the cell wall is a prerequisite for lysis.

[0022] As used herein, the term "cycle threshold" or "Ct" refers to the cycle during thermal cycling at which the increase in fluorescence due to product formation reaches a significant and detectable level above the background signal.

[0023] As used herein, the term "direct amplification" refers to a nucleic acid amplification reaction that amplifies target nucleic acid from a sample without prior purification, extraction, or concentration.This is a relative measurement of the concentration of target in a PCR reaction.Many factors other than the concentration of target affect the absolute value of Ct.Artifacts from reaction mix or equipment will change the fluorescence measurement that is related to the calculation of Ct, resulting in template-independent changes in Ct value.

[0024] As used herein, the term "extraction" refers to any action taken to remove nucleic acids from other (non-nucleic acid) materials present in a sample. Such actions include, but are not limited to, mechanical or chemical lysis, addition of detergents or proteases, or precipitation and removal of non-nucleic acids such as proteins.

[0025] As used herein, the term "interfering material" refers to any material in a sample that is not a target nucleic acid. The interfering material includes synthetic materials and biological materials. The synthetic materials include chemicals and pharmaceuticals. The biological materials include blood, urine, proteins and other biomolecules. [Brief description of the drawings]

[0026] [Figure 1] (A) is a line graph showing the results of an H1N1 assay where samples were directly amplified according to the FastStart protocol with FastStart buffer; (B) is a line graph showing the results of an H1N1 assay where samples were directly amplified according to the GoTaq protocol with GoTaq buffer. [Diagram 2] (A) and (B) are line graphs showing the effect of two sample storage buffers, universal transport medium (UTM) (A) and 1× Tris-EDTA ("TE") (B), using the FastStart protocol. [Diagram 3] (A) is a line graph showing the results of an H1N1 assay in which samples were amplified according to the FastStart protocol with FastStart Buffer after nucleic acid extraction; (B) is a line graph showing the results of an H1N1 assay in which samples were amplified directly according to the FastStart protocol with FastStart Buffer without prior nucleic acid extraction or purification. [Figure 4] (A) and (B) are line graphs demonstrating the efficacy of GoTaq chemistry and cycling conditions for direct amplification using influenza A positive samples. [Diagram 5] 1 is a line graph showing the sensitivity of a direct amplification assay with KCl compared to a direct amplification assay without KCl. [Figure 6] 1 is a line graph showing the sensitivity of a direct amplification assay with detergent compared to a direct amplification assay without detergent. [Figure 7]1 is a line graph showing the sensitivity of a direct amplification assay with preheating compared to a direct amplification assay without preheating. [Figure 8] 1 is a line graph showing amplification plots obtained from a single blood sample exposed to different anticoagulants (heparin, EDTA, citrate) in different tubes. [Figure 9] 1 is a line graph showing the ability of the direct amplification assay to detect samples obtained from buccal swabs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description The present invention relates to diagnostic methods for the detection of human pathogens including, for example, influenza A and B viruses and respiratory viruses such as respiratory syncytial virus (RSV), enteroviruses, herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, respectively), varicella zoster virus (VZV); and (pathogenic) bacteria such as Clostridium difficile, using PCR methods that do not include an extraction or purification step to isolate viral / bacterial (i.e. target) nucleic acids prior to PCR, providing substantially equivalent (or superior) sensitivity to similar assays using specific extraction or purification protocols.

[0028] More specifically, the method includes adding a detergent to the PCR mixture to dissolve cells or virions, in combination with a certain treatment step to increase the availability of target nucleic acid.Then, the sample is heated to about 50°C before the reverse transcription step, thereby promoting viral dissolution and inactivation of RNase.After the RT step, the PCR reaction is performed.Reverse transcriptase is not required in the case of bacterial or DNA virus targets.

[0029] The patient sample is added to the reagent mix at a ratio of approximately 10-40% patient sample to 60-90% reagent mix, optimally 20-30% patient sample to 70-80% reagent mix. The reagent mix contains a Thermus aquaticus derived polymerase (e.g., GoTaq Flexi DNA Polymerase™; Promega) and an amplification buffer containing an ionic detergent (e.g., GoTaq Flexi PCR Buffer™; Promega). The amplification buffer may be supplied as a 10X buffer, but is diluted to a concentration of about 5X, about 2.5X, or about 1X for use. The reagent mix further contains KCl (for viral samples only) and MgCl2, as well as dNTPs. In one formulation encompassed by the present invention, the RT-PCR reagent mix contains 0.5 μL of 5X GoTaq Flexi™ PCR buffer, 0.25 μL of 25 mM MgCl2, 0.05 μL of 10 mM dNTPs, 0.20 μL of 5 U / μL Go Taq Flexi DNA™ polymerase, and 0.5 μL of 10 mM KCl. The patient sample may be heated either before or after the RT step, and then PCR is performed.

[0030] The reaction mixture of the present invention allows for the direct amplification of nucleic acids from samples without the need for nucleic acid extraction or purification prior to amplification. Without wishing to be bound by any theory, it is believed that dissolution, when necessary, occurs through a combination of heat and detergent action. In addition, it is believed that the reagent mixture of the present invention neutralizes amplification inhibitors that are typically present in RT-PCR reactions, eliminating the need for sample dilution, a standard technique used in other direct amplification methods. The relatively high salt concentration may contribute to performance by increasing oligonucleotide binding efficiency. However, the reagent mixture will vary based on the type of target nucleic acid.

[0031] In one embodiment, the reagent mix for viral RNA detection contains at least reverse transcriptase, high concentration forward and reverse primers, optimally Scorpion primers, MgCl2, potassium chloride, dNTPs, 5X GoTaq Flexi™ PCR buffer (Promega; Cat. No. M891A or M890A) or equivalent, and 5 U / μl Taq polymerase (e.g., GoTaq Flexi DNA Polymerase™; Promega Cat. No. M8295) from Thermus aquaticus. RNAsin may be added to improve performance. Additionally, the reagent mix for pathogen detection in cerebrospinal fluid or fecal samples also contains BSA.

[0032] The reagent mixture for bacterial detection should contain at least high concentrations of forward and reverse primers, optimally Scorpion primers, MgCl2, BSA, dNTPs, 5X GoTaq Flexi™ PCR buffer (Promega; Cat. No. M891A or M890A) or equivalent, and 5 U / μl Taq polymerase (e.g., GoTaq Flexi DNA Polymerase™; Promega Cat. No. M8295) from T. aquaticus. RNAsin may be added to improve performance.

[0033] In one embodiment, the assay includes a non-template control (NTC), a positive control, and a DNA internal control (IC). The assay can be evaluated based on the Ct value of the control. In one embodiment, in an assay for detecting C. difficile, if the Ct value of the NTC is 0 and the IC is ≦40, the control is valid. In another example, in an assay for detecting C. difficile, if the Ct value of the positive control is 0, the assay is considered invalid. In another example, in an assay for detecting C. difficile, if the NTC is valid and the Ct value of C. difficile is ≦40 but ≠0, the execution of the assay is considered valid and acceptable. In another example, in an assay for detecting C. difficile, if the Ct value of C. difficile is =0 and the Ct value of IC is ≦40 but ≠0, C. difficile is considered not detected. In another example, in an assay for detecting C. difficile, if the C. difficile Ct value is =0 and the IC Ct is =0, the assay is considered invalid.

[0034] Source of viral particles Obtaining viral nucleic acid for detection assay from a sample can be done by collecting liquid samples, extracting solid or semi-solid samples, swabbing surfaces, or using additional techniques. Viral RNA can be assayed directly if the concentration present is sufficient to provide target RNA for RT-PCR reaction. Alternatively, virions can be concentrated by methods such as centrifugation, binding to surfaces by immunoadsorption or other interactions, or filtration.

[0035] Source of bacterial cells Obtaining bacterial cells for detection assay from a sample can be done by collecting liquid samples, extracting solid or semi-solid samples, swabbing a surface, or using additional techniques. Bacterial cells can be assayed directly if the concentration present is sufficient to provide target RNA for RT-PCR reaction. Alternatively, bacterial cells can be concentrated by methods such as centrifugation, binding to a surface by immunoadsorption or other interactions, or filtration. Furthermore, bacterial cell numbers can be increased by growing the cells on culture plates or in liquid medium before concentration or direct assay.

[0036] Exemplary bacteria suitable in the context of the present invention are bacteria of the genus Listeria, Escherichia, Salmonella, Campylobacter, Clostridium, Helicobacter, Mycobacterium, Staphylococcus, Campylobacter, The gram-negative and gram-positive bacteria include, but are not limited to, Enterococcus, Bacillus, Neisseria, Shigella, Streptococcus, Vibrio, Yersinia, Bordetella, Borrelia, and Pseudomonas.

[0037] target nucleic acid RNA types that can be assayed as target nucleic acids include rRNA, mRNA, transfer RNA (tRNA), or other RNA polynucleotides. rRNA species include 5S, 16S, and 23S polynucleotides that contain one or more subsequences that are unique to groups of related bacteria. The ability to detect characteristic sequences varies and depends on the level of relatedness of the virus or bacteria to be detected by the assay. Other RNA polynucleotides may be used as diagnostic target RNAs as long as they contain unique subsequences that adequately distinguish them from other bacteria at the desired level of relatedness. Examples can be found from tRNA and mRNA species, as well as from any RNA produced in bacterial cells that contains one or more characteristic subsequences. Those skilled in the art can design primers so that they initiate the synthesis of copy DNA using the target RNA as a template in a reverse transcription reaction. Also, those skilled in the art know how to design pairs of primers to amplify unique subsequences of target RNA using copy DNA as a template in PCR. It is well known to those skilled in the art that primers used synchronously in PCR must have the same hybridization melting temperature. The diagnostic target RNA within the bacterial cells must be accessible to the RT or RT-PCR reaction composition. After collection, the nucleic acid sample may be added directly to the reaction composition, which is then thermally cycled.

[0038] A specific lysis agent may be optionally present, but is preferably not added to the reagent mixture since sufficient release of viral / bacterial nucleic acid from the sample is obtained without its use. Generally, the lysis agent is added before contact with the RT, RT-PCR, or PCR reaction composition. The use of lysis agents is well known to those skilled in the art. Lysis agents include, but are not limited to, chemicals, enzymes, physical shear forces, osmotic agents, and high temperatures. The term "lysis buffer" refers to a buffer containing at least one lysis agent. Exemplary enzymatic lysis agents include, but are not limited to, lysozyme, glucolase, zymolose, lyticase, proteinase K, proteinase E, and viral endolysins and exolysins. Viral endolysins and exolysins are derived from bacteriophages or prophage bacteria and combinations thereof. Exemplary viral endolysins include, but are not limited to, endolysins from Listeria bacteriophages (A118 and PLYI18), endolysin from bacteriophage PM2, endolysin from B. subtilis bacteriophage PBSX, endolysins from Lactobacillus prophages Lj928, Lj965 and bacteriophage 15 Phiadh, endolysin from Streptococcus pneumonia bacteriophage Cp-I (Cpl-I) and the bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30. These last two have different bacterial strain specificities. Also contemplated are two-component holin-endolysins, cell lysis genes, holWMY and IysWMY of Staphylococcus wameri M phage varphiWMY. Combinations of these endolysins are also contemplated. For a discussion of viral lysis, see especially Loessner, MJ et al. (1995) Applied Environmental Microbiology I 61: 1150-1152.

[0039] Rather than using endolysins, heat treatment before or after the RT step aids in lysis in the methods of the invention. The accessibility of bacterial RNA, which is a template for RT or RT-PCR, can be improved by incubating the sample at a temperature range from about 25°C to less than about 100°C, preferably from about 50°C to 75°C. This heat pretreatment can range in time from about 1 minute to about 60 minutes, depending on the incubation temperature, with 1-20 minutes of treatment being typical. Heat treatment can include multiple incubations at different temperatures. Heat treatment can be performed in the presence or absence of RNase inhibitors, as described below. A particularly useful treatment is at about 50°C for about 5-20 minutes in the presence of RNase inhibitors.

[0040] At least one RNase inhibitor can be added to virions or bacterial cells. Typically, the inhibitor and their concentration are selected so as not to interfere with any of the primer-directed amplification processes and components. RNase inhibitors are known to those skilled in the art and include chemicals such as guanidinium isothiocyanate and diethyl-pyrocarbonate, protein inhibitors such as Superaseln (Ambion), RNase Block (Stratagene), human placental ribonuclease inhibitor and porcine liver RNase inhibitor (Takara Mirus Bio), anti-nuclease antibodies such as Anti-RNase (Novagen) and Ribonuclease Inhib III (PanVera), and reagents such as RNAlater (Ambion) and RNA protect Bacteria Reagent (Qiagen).

[0041] Assay Method In the method of the present invention, the presence of diagnostic target RNA is tested by reverse transcription alone or, preferably, by reverse transcription and polymerase chain reaction. When used together, reverse transcription and polymerase chain reaction can be performed in two steps sequentially or in one step by adding all reaction composition reagents to the sample. A DNA copy can be synthesized from the target RNA by incubating the sample in the reverse transcription reaction composition. The reagent mix contains a primer that hybridizes to the target RNA and initiates the synthesis of the copy DNA. In addition, the reagent mix contains dNTPs, MgCl2, KCl (viral samples only), reverse transcriptase and reverse transcriptase buffer (viral samples only), and, in the case of stool samples, BSA (bacterial samples only). If it is desired to create DNA copies from more than one target RNA, more than one primer may be included. However, no RNase inhibitor is used. The product of the reverse transcription reaction is then transferred to another test tube where PCR is performed according to protocols known to those skilled in the art. The PCR composition typically contains a pair of primers that initiate the synthesis of the desired DNA segment from the reverse transcribed template. In addition, PCR mixes usually contain dNTPs, a thermostable DNA polymerase such as Taq polymerase, and a polymerase buffer. If synthesis of multiple DNA segments is desired, two or more pairs of primers may be included. One new primer may be added to amplify a DNA segment with the original RT-PCR primer as the second primer of the pair. Additional reverse transcriptases that may be used for virus samples include, but are not limited to, HIV reverse transcriptase (Ambion), Transcriptor reverse transcriptase (Roche), Thermoscript reverse transcriptase (Invitrogen). Additional DNA polymerases that may be used include, but are not limited to, Pfu, Vent, and Sequitherm DNA polymerase (EPICENTRE).

[0042] Whether RT-PCR is performed in two steps or in one step, the RT step is performed first and typically consists of a single temperature incubation at a temperature between about 37°C and about 70°C. As known to those skilled in the art, different temperatures are appropriate for different Rt enzymes and different primers. The PCR reaction that follows typically consists of an initial incubation at about 94°C to about 96°C for about 6 to about 15 minutes. This step is used to denature the cDNA and activate the heat-activated Taq polymerase enzyme. Multiple cycles of amplification of the cDNA target are then performed. Each cycle involves three operations: target denaturation, primer annealing, and primer extension. Target denaturation typically occurs at a temperature greater than about 90°C. Primer annealing temperature is defined by the melting temperature of the particular primers used in the reaction, and primer extension is performed at a temperature ranging from about 60°C to about 72°C depending on the thermostable polymerase used. If primer annealing and extension are performed at the same temperature, this is a two-temperature PCR, whereas in three-temperature PCR, each of the three steps occurs at a different temperature. After the amplification phase is complete, a final extension time is typically added to ensure synthesis of all amplification products.

[0043] Target nucleic acids also include DNA, including DNA from bacterial species and DNA viruses, for example. Viral DNA suitable for evaluation includes both DNA obtained directly from the viral capsid as well as DNA integrated into the genome of the host.

[0044] Detection of RT and RT-PCR products Methods for directly detecting the cDNA products of RT reactions are known to those skilled in the art and use labels incorporated into or attached to the cDNA products. Signal generating labels that can be used are known to those skilled in the art and include, for example, fluorescent moieties, chemiluminescent moieties, particles, enzymes, radioactive tags, or luminescent moieties or molecules. Fluorescent moieties are particularly useful, especially fluorescent dyes that can be attached to nucleic acids and emit fluorescent signals. Various dyes are known to those skilled in the art, such as fluorescein, Texas Red, and rhodamine. Particularly useful are the monoreactive dyes Cy3 and Cy5, both of which are commercially available (e.g., from Amersham Pharmacia Biotech, Arlington Heights, Ill.). A more sensitive method for specifically detecting labeled DNA is to hybridize the products to target DNA sequence molecules immobilized in a matrix such as a nylon membrane or glass slide. The signal after hybridization can then be scanned by a laser scanner using appropriate filtering to detect the particular dye used. This is known to those skilled in the art, especially in DNA microarray technology. The label may be incorporated into the cDNA during synthesis of the cDNA in the RT reaction, or the label may be attached to the cDNA product after synthesis of the cDNA. For example, the RT reaction can be performed with a labeled primer. One type of labeled primer has a particle attached with multiple signal generating molecules. Reverse transcription using labeled nucleotides such as dye-labeled UTP and / or CTP incorporates the label into the transcribed nucleic acid. Alternatively, a post-synthesis coupling reaction can be used to detect the cDNA product. Attachment of labels to nucleic acids is known to those skilled in the art and can be done, for example, by end-labeling with, for example, labeled RNA, or by treating the nucleic acid with a kinase and then attaching a nucleic acid linker to link the sample nucleic acid with a label (e.g., a fluorophore). In another labeling method, the DNA product from the RT reaction is amplified by combining it with an in vitro transcription reaction. For example, a T7 promoter region is incorporated into the primer used for the RT reaction.A T7 in vitro transcription kit is then used to generate large amounts of RNA to increase the sensitivity of detection, which can be purchased from Ambion (2130 Woodward, Austin, Tex.) or other commercial suppliers.

[0045] RT-PCR detection Methods for detecting RT-PCR products include separation by gel electrolysis and ethidium bromide staining, or detection of fluorescent or radioactive labels incorporated into the products. Methods that do not require a step of separating the amplification products prior to detection can also be used. These methods are generally called real-time PCR or homogeneous detection. Most real-time methods detect the formation of amplification products by monitoring the change in fluorescence during thermal cycling. These methods include, but are not limited to, TaqMan® dual-labeled probes (Applied Biosystems, Foster City, Calif. 94404), Molecular Beacons (Tyagi S and Kramer FR (1996) Nat BiotechnoI14:303-308), and SYBR® Green dye (Molecular Probes, Inc Eugene, Oreg. 97402-0469). Some of these same homogeneous methods can also be used for end-point detection of amplification products. An example of this type of method is SYBR® Green dye dissociation curve analysis. In melting curve analysis, a final slow ramp, typically at temperatures between about 60°C and 90°C, combined with fluorescence monitoring allows detection of the melting point and thus the presence of amplification product (Ririe et al. (1997) Anal. Biochem. 245: 154-60).

[0046] Assay Sensitivity The sensitivity of a direct amplification assay can be increased by adding one or more sensitivity enhancing components to the buffer used in the assay. Such components include, but are not limited to, KCl, a detergent and albumin. In some embodiments, the albumin is bovine serum albumin. In some embodiments, the detergent is a cationic detergent. The sensitivity of a direct amplification assay can also be increased by providing additional heat to the assay, such as pre-heating the sample before adding the reagent. In some embodiments, the sensitivity can be increased by a combination of a sensitivity enhancing component and additional heat. EXAMPLES

[0047] The methods of the present invention have now been generally described, but will be more readily understood by reference to the following examples, which are provided for illustrative purposes and are not intended to limit the methods and kits of the present invention.

[0048] Example 1: Universal Master Mix Unless otherwise specified, a 2.5X Universal Master Mix (UMM) is prepared in the following ratios: The table below shows suitable volumes of reagents to prepare 15 ml of UMM, however, any suitable volumes can be prepared as needed. [Table 1]

[0049] GoTaq Flexi Buffer™ and its equivalents contain an ionic detergent and are magnesium-free.

[0050] Example 2: Effect of KCl on direct amplification of respiratory viral nucleic acids Artificially engineered swab specimens containing influenza B virus were prepared by spiking cultured influenza B virus ("Flu B") (Great Lakes strain) with an internal control nucleic acid into the viral transport medium samples. Ct values ​​for Flu B and the control nucleic acid were measured in the presence and absence of 25 mM KCl using the universal master mix of Example 1, which contains UMM minus KCl and either 1 or 2 μl of reverse transcriptase (Improm II reverse transcriptase, Promega catalog number A3800). Serial dilutions of template copies of Flu B virus were assayed at TCID50 of 158 and 39.5. 50 The RT-PCR reactions were carried out as follows.

[0051] Step 1: 75°C for 3 minutes (1 time) Step 2: 47°C for 10 minutes (1 time) Step 3: 97°C for 2 minutes (1 time) Step 4: 102° C. for 1 second, then 60° C. for 20 seconds for data collection (repeated 45 times).

[0052] The threshold for Ct measurement was 50,000. The fluorophores for Flu B and the internal control probe were JOE and Q670, respectively. All assays were performed in duplicate and the results were averaged. [Table 2] [Table 3]

[0053] The results in Tables 1 and 2 demonstrate that the presence of KCl increases the sensitivity of a direct RT-PCR amplification assay for respiratory viruses (Flu B) in serum at low virus concentrations. These results further indicate that the presence of KCl reduces or negates the need to concentrate and / or purify virus from serum prior to analysis by RT-PCR.

[0054] Example 3: Direct Amplification of Respiratory Viral Nucleic Acid from Clinical Samples Various clinical samples (oral swabs and cerebrospinal fluid) were evaluated for the presence of HSV-1 and / or HSV-2. The universal MM of Example 1 was used with noted modifications for MgCl2 and KCl.

[0055] The oral swab RT-PCR amplification master mix was as shown in the table below. [Table 4]

[0056] The CSF RT-PCR amplification master mix was as shown in the table below. [Table 5]

[0057] Clinical samples were analyzed using both direct RT-PCR amplification protocols and RT-PCR amplification protocols that first use a nucleic acid extraction protocol. Nucleic acids were extracted using a Roche MagNA Pure LC instrument and the corresponding Total Nucleic Acid Isolation Kit. A total of 200 μl of sample was extracted and nucleic acids were eluted in 50 μl.

[0058] For each assay, 10 μl of sample was added to 40 μl of the master mix described in this example. RT-PCR reactions were performed as follows.

[0059] Step 1: 75°C for 3 minutes (1 time) Step 2: 47°C for 10 minutes (1 time) Step 3: 97°C for 2 minutes (1 time) Step 4: 102° C. for 1 second, then 60° C. for 10 seconds for data collection (50 repeats).

[0060] The results are as follows: [Table 6]

[0061] These data demonstrate that for HSV-infected oral or CSF samples, the direct amplification method described above gives equivalent results in a multiplex RT-PCR amplification assay compared to an RT-PCR protocol that involves nucleic acid extraction and concentration prior to analysis.

[0062] Example 4: Effect of RNAse inhibitors on direct nucleic acid amplification assays Control virus was added to the viral transport medium to create synthetic samples for analysis using the direct amplification RT-PCR assay described above. In each assay, 10 μl of sample was added to 40 μl of UMM described in Example 1 in the presence or absence of 1 μl of RNAse inhibitor ("RNAsin") (Promega catalog number N261B). RT-PCR reactions were performed as follows.

[0063] Step 1: 50°C for 10 minutes (1 time) Step 2: 97°C for 2 minutes (1 time) Step 3: 102° C. for 1 second, then 58° C. for 20 seconds for data collection (50 repeats).

[0064] In the absence of RNAsin, the Ct could not be measured, suggesting that the viral RNA was degraded prior to RT. Assays performed in duplicate yielded an average Ct of 32.5 in the presence of RNAsin. These results demonstrate that the presence of RNAsin improves the sensitivity of the direct RT-PCR amplification method.

[0065] Example 5: Effect of sample pre-heating on direct nucleic acid amplification assays A combination of influenza A, influenza B, and RSV viruses was added to the viral transport medium at approximately 5,000 viral copies / ml to form the "FABR" synthetic sample, or influenza B virus (10 -4) alone was added. The samples were evaluated by direct amplification using the universal master mix of Example 1 plus 1 μl Improm II reverse transcriptase and 0.25 μl RNAsin. MS2 phage was added as an internal control. Experimental samples were preheated to 75° C. for 3 minutes and 10 μl of sample was added to 40 μl of universal master mix for each assay. RT-PCR reactions were performed as follows:

[0066] Step 1: 50°C for 10 minutes (1 time) Step 2: 97°C for 2 minutes (1 time) Step 3: 102° C. for 1 second, then 58° C. for 20 seconds for data collection (50 repeats).

[0067] The results are as follows: [Table 7]

[0068] Next, control cerebrospinal fluid samples were diluted with HSV-1 virus (TCID 50 Control viruses were evaluated in the absence of RNAsin, with and without sample preheating, with the addition of 0.1% ribosomal RNA (100 / ml = 2.14). The specificity of the HSV-1 detection method was confirmed by simultaneously evaluating the samples for the presence of HSV-2 nucleic acid. No HSV-2 was detected in any of the samples. The results are as follows: [Table 8]

[0069] These results demonstrate that brief preheating of samples prior to direct RT-PCR amplification and evaluation increases the sensitivity of virus detection in many cases (i.e., at least for FluB, RSV, and HSV-1) and does not negatively affect sensitivity in other cases (i.e., FluA). Furthermore, preheating of samples reduces or eliminates the need for the addition of RNase inhibitors.

[0070] Example 6A: Stool Sample Protocol Using BSA Human stool samples are obtained from patients using standard clinical methods. Samples are kept at 2-25°C for transport and short-term storage, with no more than two freeze / thaw cycles prior to use. For evaluation, a flocked swab is dipped into the thoroughly mixed stool specimen and excess stool is removed by pressing the swab against the side of the specimen container. The swab is then swirled in 1 ml of Tris-EDTA (TE) buffer and discarded. The sample is heated to 97°C for 10 minutes. The sample is then diluted 1:4 with the UMM of Example 1 (with modifications of the UMM described below) (i.e., add 2 μl of sample to 8 μl of UMM). Optionally, 2 μl of solution containing a positive internal control nucleic acid can be added to the 8 μl of UMM (containing 4 μl of UMM, as well as 0.35 mg / ml of BSA, 600 nM each of forward and reverse primers, 300 nM each of internal control primers, and 0.5 μl of internal control DNA). The C. difficile target primers were labeled with a FAM fluorophore and the internal control target was labeled with a Quasar 670 fluorophore. Thermal cycling began with an initial denaturation step at 97°C for 2 min, followed by 40 cycles of 97°C for 10 s and 60°C for 30 s. Real-time PCR was performed for 40 cycles and amplification curves were measured using a fluorescently labeled probe specific for the target nucleic acid, which in this experiment was the C. difficile TCD-B gene. [Table 9]

[0071] Example 6B: Stool sample protocol without BSA To determine whether the addition of BSA reduces the inhibition of detection provided by stool material, RT-PCR assays were performed using Clostridium difficile DNA as target in both the presence and absence of BSA, following the protocol used to test samples containing BSA. Stool samples were prepared according to the recipe shown in Example 6A, except that 100X BSA was not added to the samples without BSA. The MagnaPure system (Roche) was used for purification of nucleic acids. The PCR formulation without BSA was plated in wells 1-20 and the PCR formulation with BSA was plated in wells 21-40, and the PCR reaction was performed as described above.

[0072] The results, shown in Table 6, indicate that the addition of BSA reduces the inhibition of Gram-positive anaerobic nucleic acid detection from stool samples.

[0073] Table 6: [Table 10]

[0074] Example 7: Direct RT-PCR amplification is buffer dependent Influenza A samples and internal controls were evaluated using direct amplification to determine whether the enzymes used in the universal master mix defined in Example 1 were unique in their ability to facilitate a direct detection assay. Hybrid primer concentrations were used consisting of 600 nM influenza A scorpion / primer (against the matrix gene), 500 nM porcine H1 scorpion / primer (against the hemaglutanin gene), and 150 nM armored RNA internal control (IC) scorpion primer specific for MS2 phage. Reaction mixes were prepared using two separate enzymes and buffer systems: 5X PCR buffer (Promega; Cat. No. M891A or M890A), a component of GoTaq® Flexi and its associated universal MM, and 10x buffer (Roche), a component of FastStart High Fidelity and its associated RNA MM, at the following concentrations: [Table 11]

[0075] The samples consisted of influenza virus spiked in viral transport medium. A total of 2 μl of sample was added to 8 μl of each of the reaction mixes listed above. Each sample was amplified directly without prior extraction.

[0076] Thermal cycling was then performed and amplification curves were measured using fluorescently labeled probes specific for the target nucleic acid. The InfA M gene probe, the H1N1 specific HA gene probe, and the IC probe were labeled with FAM, CFR610, and Q670, respectively. RT-PCR (ICy / Universal 96-well disk; 3M) was then performed using two cycle protocols: first, GoTaq and FastStart assays were run with GoTaq cycles, and then both assays were run with FastStart cycles. The FastStart protocol is as follows:

[0077] Step 1: 15 min at 47°C Step 2: 97°C for 10 minutes (1 time) Step 3: 97° C. for 15 sec, then 60° C. for 30 sec (repeated 40 times).

[0078] The GoTaq cycling protocol is as follows:

[0079] Step 1: 10 min at 47°C Step 2: 97°C for 2 minutes (1 time) Step 3: 97°C for 5 seconds, then 58°C for 30 seconds (repeated 40 times).

[0080] The results are shown in Figure 1(A) and (B), which show samples run using the FastStart cycling protocol with FastStart buffer and samples run using the GoTaq cycling protocol with GoTaq buffer. No amplification was observed using the FastStart cycling conditions and buffer system, whereas robust amplification was observed using the GoTaq cycling conditions and buffer system.

[0081] The effect of sample storage buffers was also compared. In particular, H1N1 nucleic acid amplification from samples stored in Universal Transport Medium (UTM) or 1X Tris-EDTA ("TE") was compared using the FastStart protocol. As shown in Figure 2, no significant amplification was observed in the UTM samples, whereas the TE samples produced robust amplification curves.

[0082] The effect of nucleic acid extraction on FastStart chemistry and cycling conditions was studied. As shown in Figure 3, it was confirmed that the FastStart protocol was unable to amplify H1N1 nucleic acids directly from clinical samples (Figure 3B). However, strong amplification was observed in samples in which the nucleic acid was extracted prior to the amplification reaction (Figure 3A). These results demonstrate that not all RT-PCR amplification conditions can be applied to a direct amplification / detection system, and that the GoTaq chemistry is particularly well suited to this assay format.

[0083] The validity of the GoTaq chemistry and cycling conditions for direct amplification was confirmed using influenza A-positive patient samples, including 2009 pandemic H1N1 positive samples. Amplification was performed using the following parameters: [Table 12]

[0084] The cycle conditions were as follows:

[0085] Step 1: 10 min at 47°C Step 2: 97°C for 2 minutes (1 time) Step 3: 97°C for 5 seconds, then 58°C for 30 seconds (repeated 40 times).

[0086] The results shown in Figure 4A are from a sample containing non-pandemic influenza A virus, and amplification of the FAM target indicates detection of influenza A, but not pandemic H1N1 influenza A. Figure 4B shows amplification of a pandemic H1N1 sample, demonstrating amplification of the influenza A target in addition to the H1N1-specific target.

[0087] Example 8: Detection of Flu A, Flu B and RSV by direct nucleic acid amplification assays and comparison with methods using nucleic acid extraction Nucleic acid from clinical specimens (swabs) and control samples was amplified using a direct nucleic acid amplification assay and the results were compared to amplification results using a method involving nucleic acid extraction. The sequences of the amplification primers are shown in the table below. [Table 13]

[0088] Clinical and control specimens were heated to 65° C.-70° C. for 5 minutes. Reaction mixtures were prepared as follows:

[0089] Reaction mixture [Table 14]

[0090] Thermal cycling was then performed using the following cycle parameters:

[0091] Cycle Parameters [Table 15]

[0092] As shown below, amplification results using a direct nucleic acid amplification assay were compared to amplification results obtained using a method involving nucleic acid extraction.

[0093] Clinical specimens (swabs) tested [Table 16]

[0094] There was 100% concordance between the results obtained using the direct nucleic acid amplification assay and the method involving nucleic acid extraction shown above.

[0095] Example 9: Detection of HSV-1, HSV-2, and VZV by direct nucleic acid amplification assays and comparison with methods using nucleic acid extraction Nucleic acids from clinical specimens as well as artificial samples were amplified using a direct nucleic acid amplification assay and the results were compared to amplification results using a method involving nucleic acid extraction. The reaction mixture was prepared as follows.

[0096] Reaction mixture [Table 17]

[0097] The sequences of the amplification primers are shown in the table below.

[0098] array [Table 18]

[0099] Thermal cycling was performed using the following cycle parameters:

[0100] Cycle Parameters [Table 19]

[0101] As shown below, amplification results using a direct nucleic acid amplification assay were compared to amplification results obtained using a method involving nucleic acid extraction.

[0102] VZV A total of 32 of 32 specimens (13 swabs, 2 vitreous fluid, and 17 CSF) were detected as positive for VZV using an amplification method that included nucleic acid extraction, while 31 of 32 were detected as positive by direct amplification. The CSF sample that tested negative was detected to have a Ct of 37.1 when tested with the addition of nuclease inhibitors.

[0103] HSV-1 and HSV-2 Clinical specimens (swabs): Two HSV-1 samples that tested positive using the amplification method that included nucleic acid extraction were also positive using the direct amplification method, and similarly, two HSV-2 samples that tested positive using the amplification method that included nucleic acid extraction were also positive using the direct amplification method.

[0104] Artificial samples: The detection results for HSV-1 and HSV-2, comparing direct amplification and nucleic acid extraction prior to amplification using artificial samples, are shown below.

[0105] HSV-2 [Table 20]

[0106] * TCID 50 :50% tissue culture infectious dose HSV-1 [Table 21]

[0107] * TCID 50 :50% tissue culture infectious dose As noted above, results using direct amplification and methods using nucleic acid extraction are comparable for the detection of HSV-1 and HSV-2.

[0108] Example 10: Detection of enteroviruses by direct nucleic acid amplification assays and comparison with methods using nucleic acid extraction Nucleic acid from the samples was amplified using a direct nucleic acid amplification assay and the results were compared to amplification results using a method involving nucleic acid extraction. The reaction mixture was prepared as follows.

[0109] Reaction mixture [Table 22]

[0110] The sequences of the amplification primers are shown in the table below.

[0111] array [Table 23]

[0112] Thermal cycling was performed using the following cycle parameters:

[0113] Cycle Parameters [Table 24]

[0114] As shown below, amplification results using a direct nucleic acid amplification assay were compared to amplification results obtained using a method involving nucleic acid extraction.

[0115] Thirty-two samples that tested negative by amplification with nucleic acid extraction were also negative by direct amplification, and 16 samples that tested positive by amplification with nucleic acid extraction were also positive by direct amplification.

[0116] The detection results comparing direct amplification and nucleic acid extraction prior to amplification using artificial samples are shown below. [Table 25]

[0117] * TCID 50 :50% tissue culture infectious dose As noted above, results using direct amplification and methods using nucleic acid extraction are comparable in detecting enteroviruses.

[0118] Example 11: Detection of Clostridium difficile by direct nucleic acid amplification assay and comparison with methods using nucleic acid extraction A direct nucleic acid amplification assay was used to amplify nucleic acid from the samples and the results were compared to amplification results using a method involving nucleic acid extraction.

[0119] Stool specimens were obtained from different individuals. A flocked swab was dipped into the stool specimen. Excess stool specimen was removed. The swab was swirled in 1 ml TE buffer and the swab was discarded. The samples were heated to 97°C in a heating block for 10 minutes.

[0120] The PCR master mix was prepared as follows.

[0121] PCR mix [Table 26]

[0122] 2 μl of heated sample was added to 8 μl of master mix and PCR was performed using the following cycle parameters: [Table 27]

[0123] The primers target the toxin B region of C. difficile. The sequences of the amplification primers and amplicon are shown below.

[0124] Clostridium difficile scorpion primer: JPEG2024081792000029.jpg11154

[0125] Clostridium difficile reverse primer: JPEG2024081792000030.jpg593

[0126] Clostridium difficile amplicon: JPEG2024081792000031.jpg14158

[0127] Fluorescent signals from the Clostridium difficile scorpion primers were detected at 495 nm, and signals from the internal control were detected at 644 nm.

[0128] As shown below, amplification results using the direct nucleic acid amplification assay were compared to results obtained using an amplification method that includes nucleic acid extraction. [Table 28]

[0129] As noted above, 99% of samples confirmed as positive by amplification with nucleic acid extraction were also confirmed as positive by the direct amplification assay, and 91% of samples confirmed as negative by amplification with nucleic acid extraction were also confirmed as negative by the direct amplification assay.

[0130] The limit of detection (LoD) was determined using a panel consisting of artificial samples in stool-TE buffer matrix spiked with C. difficile bacterial stock. The panel included samples that were negative (unspiked matrix) and at various concentrations close to the approximate LoD (obtained in the previous phase of testing). Results from 24 replicates from three separate preparations and PCR runs (8 replicates / run) at each level were analyzed by Probit Analysis to determine the lowest concentration that could be accurately detected with a 95% probability. The limit of detection is 0.04 cfu / reaction.

[0131] Assay reproducibility A reproducibility study was performed using artificial samples in stool-TE buffer matrix spiked with Clostridium difficile bacterial stock. The panel included negative (no spiked matrix), low positive (approximately 2-4x LOD), and moderate positive (approximately 8-10x LOD) samples. The reproducibility study was performed over 5 days (not consecutive days) using two Integrated Cycler instruments. On each day, two runs were performed on each instrument. Each run included four replicates of each panel member and positive control (PC) and one replicate of no template control (NTC). In each run of the Integrated Cycler instrument, the panel and PC were assayed in quadruplicate and the NTC was assayed once. The panel was run over 5 days (not consecutive days) with two runs per instrument per day using one lot of direct amplification assay. There was a minimum of two instruments with at least one operator per instrument. A summary of the reproducibility of the results is shown below. [Table 29]

[0132] Assay performance in the presence of potential interfering substances The performance of the assay was evaluated with the addition of potential interfering substances that may be present in stool samples at the concentrations shown in the table below. A total of 21 potential interfering substances (each with four replicates) and baseline (positive) samples (five replicates) were initially tested. All but two interfering samples were scored as "positive" in all four replicates in the initial run. For two interfering substances (Vancomycin and Pepto-Bismol), all five replicates were "positive" in the second confirmatory run. No interference was observed. [Table 30]

[0133] Cross-reactivity Analytical specificity was performed for a variety of possible cross-reactants. A total of 47 potential cross-reacting organisms were tested. Only the Rotavirus organism was scored as "negative" in all three replicates in the initial test. Five replicate confirmatory runs for "Rotavirus" were also scored as "negative." No cross-reactivity was identified. [Table 31] JPEG2024081792000036.jpg255163JPEG2024081792000037.jpg170163

[0134] * Subsequent testing of this sample in a clinical testing laboratory confirmed that the sample was positive for Clostridium difficile.

[0135] Example 12: Detection of Group A Streptococcus by Direct Nucleic Acid Amplification Assay and Comparison with Methods Using Nucleic Acid Extraction Nucleic acid from the samples was amplified using a direct nucleic acid amplification assay and the results were compared to amplification results using a method involving nucleic acid extraction.

[0136] The swab samples were used in a direct amplification assay. The PCR master mix was prepared as follows:

[0137] PCR mix [Table 32]

[0138] 2 μL of transport medium from the swab sample was added to 8 μL of PCR master mix and PCR was performed using the following cycle parameters: [Table 33]

[0139] The sequences of the amplification primers and amplicons are shown below.

[0140] Group A Streptococcus Scorpion Primer: JPEG2024081792000040.jpg17153

[0141] Group A Streptococcus reverse primer: JPEG2024081792000041.jpg877

[0142] Group A Streptococcus amplicon: JPEG2024081792000042.jpg14156

[0143] As shown below, amplification results using a direct nucleic acid amplification assay were compared to results obtained using an amplification method that includes nucleic acid extraction. [Table 34]

[0144] As noted above, 93.8% of samples confirmed as positive by amplification with nucleic acid extraction were also confirmed as positive by the direct amplification assay, and 99% of samples confirmed as negative by amplification with nucleic acid extraction were also confirmed as negative by the direct amplification assay.

[0145] Example 13: Detection of Flu A, Flu B and RSV by direct nucleic acid amplification assays in the presence of interfering substances Using the experimental protocol of Example 8, nucleic acid from clinical specimens was amplified using a direct nucleic acid amplification assay in the presence of a variety of interfering substances, as listed in the table below. [Table 35]

[0146] Direct amplification assays were performed in duplicate for each potential interferent. Q670 fluorescent label was used as an internal control, PC represents the positive control, and NEG represents the negative control.

[0147] The table below presents the Ct results for Flu A virus detection in the presence of potential interfering substances. [Table 36]

[0148] The table below presents the Ct results for Flu B virus detection in the presence of potential interfering substances. [Table 37]

[0149] The table below presents the Ct results for RSV virus detection in the presence of potential interfering substances. [Table 38]

[0150] *A false positive FAM signal was detected in one of two replicates. Results were confirmed to be accurate based on the negative control having no significant amplified signal in the FAM, JOE or CFR610 channels and a Ct < 40 in the Q670 channel, and the PC reaction gave a Ct < 40 in the FAM, JOE and CFR610 channels.

[0151] The results of the direct amplification assay for detection of Flu A, Flu B and RSV demonstrate that for any of the viruses tested, there was no significant change in Ct value when any of the interfering substances were used compared to the interference-free control samples. Thus, the direct amplification assay is not affected by potential interfering substances when detecting low positive samples of these viruses.

[0152] Example 14: Detection of HSV-1 and HSV-2 by direct nucleic acid amplification assay in the presence of interfering substances Using the experimental protocol of Example 3, nucleic acids on the negative swab matrix and in synthetic cerebrospinal fluid were amplified using a direct nucleic acid amplification assay in the presence of various interfering substances listed in the table below. [Table 39]

[0153] A potential whole blood interferent (interferent ID: 1) was tested at 10%, which is more clinically relevant than purified hemoglobin, and a potential hemoglobin interferent (interferent ID: 9) was tested at higher concentrations, from 5.0 to 1.25 mg / mL, which inhibited the detection of HSV-1 and HSV-2.

[0154] Direct amplification assays were performed in triplicate for each potential interfering substance. Q670 fluorescent label was used as an internal control, PC represents the positive control, and NEG represents the negative control.

[0155] The table below presents the Ct results for HSV-1 virus detection on a negative swab matrix in the presence of potential interfering substances. [Table 40]

[0156] Although HSV-1 was detected in the female urine sample, the liquid check for female urine (interferent ID: 2) failed as indicated by a higher fluorescence value than the no sample control. KY brand jelly (interferent ID: 5) gave an earlier Ct value compared to the control sample, even though the same HSV-1 level was used for all potential interferent tests. The female urine and KY jelly potential interferents were retested and the earlier Cts were reproduced for both samples.

[0157] The table below presents the Ct results for HSV-2 virus detection on a negative swab matrix in the presence of potential interfering substances. [Table 41]

[0158] The problems with liquid checks on female urine samples for HSV-1 described above also applied to HSV-2.

[0159] The table below presents the Ct results for HSV-1 virus detection in synthetic cerebrospinal fluid in the presence of potential interfering substances. [Table 42]

[0160] The table below presents the Ct results for HSV-2 virus detection in synthetic cerebrospinal fluid in the presence of potential interfering substances. [Table 43]

[0161] Results were confirmed to be accurate based on the negative control having no significant amplified signal in the FAM or CFR610 channels and a Ct < 40 in the Q670 channel, and the PC response gave a Ct < 40 in the FAM and CFR610 channels.

[0162] The results of the direct amplification assay for the detection of HSV-1 and HSV-2 demonstrate that there was no significant change in Ct value with any of the interfering substances for either virus tested, therefore the direct amplification assay is not affected by potential interfering substances when detecting low positive samples for these viruses.

[0163] Example 15: Components of a direct amplification assay that provide improved performance The data below represents the components of the direct amplification assay that provide improved performance. In one embodiment, all of these components are combined to provide a system that allows for efficient real-time PCR even in the presence of known inhibitors (e.g., blood and heparin).

[0164] Effect of KCl Addition of KCl (up to 20-40 mM) to the direct amplification assay improves the fluorescent signal, giving the reaction improved sensitivity. The data in Figure 5 show detection of MRSA in the presence and absence of KCl. Although some detection is possible in the absence of KCl, the presence of KCl improves the effectiveness of the reaction. The data in Figure 5 were obtained in the presence of other reaction components such as a cationic surfactant.

[0165] Effects of BSA BSA was added at various concentrations to stool samples containing C. difficile. At the higher concentration, represented below as 3500 ng / reaction, inhibition was removed from some patient samples. As can be seen in the table below (representing Ct values), at the lower BSA concentration of 5 ng / reaction, the C. difficile target was not detected and the internal control was not detected in two out of three samples. Specifically, the internal control was detected using sample A. However, compared to the higher concentration samples, the lower Ct values ​​evidence slower amplification, characteristic of inhibition (Ct values ​​are the PCR cycles at which sufficient signal is produced to detect the target in question). [Table 44]

[0166] Effect of surfactants The addition of cationic detergent improves the fluorescent signal, giving improved sensitivity to the direct amplification assay. As shown in Figure 6, some detection is possible when performing a direct amplification assay for Simplexa Bordetella without the addition of detergent. However, the use of detergent improves the efficiency of the reaction, as shown by the higher signal height, thereby resulting in improved sensitivity.

[0167] Effect of additional heating Some organisms tested require an additional heating step to improve assay performance. For example, as shown in FIG. 7, improved sensitivity was observed for Flu B when additional heating beyond that provided in the standard reaction (e.g., preheating) was used. Improvement with Flu B was seen at a temperature of 70° C. Performance of the assay to detect other organisms, such as Clostridium difficile and Group A Streptococcus, was seen at a temperature of 95° C. Heating the sample to destroy inhibitors and lyse the organism must be balanced against the possibility that heat will destroy the reagents. In some embodiments, heating the sample is done before adding the reagents (e.g., buffers).

[0168] System tolerance The data below (Bordetella pertussis / parapertussis PCR) shows that the direct amplification assay can tolerate up to 30% transport medium (Copan UTM) without inhibition. All samples tested by the direct detection method below used 30% sample and 70% direct amplification reaction mix. Direct detection results were compared to results using DNA extraction and purification prior to amplification. When using patient specimens, the direct method had 99% sensitivity and specificity compared to the extraction and purification test. [Table 45]

[0169] Previous publications have limited sensitivity because they required significant dilution of the sample before adding it to the reaction mixture.

[0170] Example 16: Direct Amplification Assays with Additional Sample Types whole blood Human genetic testing was performed using whole blood. Whole blood could be used either at a 1:4 dilution or at 0.5 μl in a 10 μl reaction volume. In either case, heme (a known PCR inhibitor) did not affect the assay. Complete concordance was achieved when testing for the presence of factor V Leiden or factor II mutations using the direct amplification method versus the standard method utilizing nucleic acid extraction prior to amplification and mutation detection. [Table 46]

[0171] Heparinized whole blood The data in Figure 8 shows amplification plots from one blood sample collected in three tubes, each with a different anticoagulant (heparin, EDTA, citrate). As shown in the figure, the amplification plots show that all samples give effective amplification, even when heparin, a known PCR inhibitor, is used as the anticoagulant.

[0172] Oral swab The data in Figure 9 represents four replicates of two samples of human genomic DNA and one negative control for mutations (detection of the Factor V Leiden mutation region). Efficient amplification was observed in all replicates. Samples were collected by swabbing the inside of the cheek for approximately 10 seconds, ensuring that the entire swab surface was used. The swab was then placed in 500 μL of 2 μL of 1X TE buffer, which was placed directly into the PCR sample without extraction.

[0173] Comparison with previously published literature Previously published results indicate that samples must be diluted for effective detection. Compared to these publications, e.g., reference Pandori et al., BMC Infect. Dis., 6:104 (2006), the data above demonstrate that 10 times the amount of sample provides a detection limit 10 times lower than the published method.

[0174] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0175] The invention illustratively described herein may be suitably practiced in the absence of any element(s), limitation(s) not specifically disclosed herein. Moreover, the terms and expressions used herein are used as terms of description and not of limitation, and there is no intention in the use of said terms and expressions to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention described in the claims. Thus, although the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the invention embodied therein disclosed herein may be practiced by those skilled in the art, and such modifications and variations are deemed to be within the scope of the present invention.

[0176] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes any negative limitation that removes any subject matter from the generic description or genus of the invention with a condition (whether or not the omitted material is specifically recited herein).

[0177] Other embodiments are within the scope of the following claims. Furthermore, when features or aspects of the invention are described in terms of Markush groups, those skilled in the art will thereby recognize that the invention can also be described in terms of any individual members or subgroups of members of the Markush group.

[0178] The present invention also provides the following aspects. [1] A method for confirming the presence or absence of a target nucleic acid derived from a microorganism in a biological sample obtained from a human, the method comprising: (a) contacting the sample with a DNA polymerase and a buffer under conditions suitable for amplification of a target nucleic acid from the sample without extracting the target nucleic acid from the sample; (b) subjecting the sample obtained by step (a) to thermal cycling so as to amplify the target nucleic acid, if present; and (c) detecting the amplified target nucleic acid produced by step (b), if present. Including, The nucleic acids in the sample are not extracted from the sample prior to amplification; The method. [2] The method according to [1], wherein the sample is not diluted prior to step (a). [3] The method according to [1], wherein the sample is heated prior to step (b). [4] The method according to [3], wherein the sample is heated prior to step (a). [5] The method according to [1], wherein the sample is heated to a temperature of about 70°C or higher for about 2 minutes or more prior to step (b). [6] The method according to [1], wherein the buffer contains potassium chloride (KCl). [7] The method according to [6], wherein KCl is present at a concentration of about 5 mM to about 50 mM. [8] The method of [1], wherein the buffer comprises GoTaq(TM) Flexi buffer. [9] The method of [8], wherein GoTaq™ Flexi Buffer is present at a concentration of 1X to 5X during step (b).

[10] The method according to [1], wherein the DNA polymerase is Taq polymerase.

[11] The method according to [1], wherein the target nucleic acid is DNA.

[12] The method according to [1], wherein the target nucleic acid is RNA.

[13] The method of

[12] , further comprising contacting the sample with a reverse transcriptase prior to amplification.

[14] The method according to

[13] , wherein the sample is contacted with DNA polymerase and reverse transcriptase simultaneously.

[15] The method according to [1], wherein the sample is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, oral fluid, and stool.

[16] The method according to

[15] , wherein the blood is whole blood.

[17] The method of [1], wherein the sample is obtained from the oral cavity region.

[18] The method according to [1], wherein the microorganism is a virus.

[19] The method according to

[18] , wherein the virus is selected from the group consisting of influenza virus, respiratory syncytial virus, varicella zoster virus, herpes simplex virus, and enterovirus.

[20] The method according to [1], wherein the microorganism is a bacterium.

[21] The method according to

[20] , wherein the bacterium is of the genus Clostridium or Streptococcus.

[22] The method according to [1], wherein the buffer further contains albumin.

[23] The method according to

[22] , wherein the albumin is BSA.

[24] The method according to [1], wherein the buffer further contains a surfactant.

[25] The method according to

[24] , wherein the surfactant is a cationic surfactant.

[26] A method for confirming the presence or absence of a target nucleic acid derived from a microorganism in a biological sample obtained from a human, the method comprising: (a) contacting the sample with a DNA polymerase and a buffer under conditions suitable for amplification of a target nucleic acid from the sample without extracting the target nucleic acid from the sample; (b) subjecting the sample obtained by step (a) to thermal cycling so as to amplify the target nucleic acid, if present; and (c) detecting the amplified target nucleic acid produced by step (b), if present. Including, The nucleic acid in the sample is not extracted from the sample prior to amplification, and The buffer solution contains at least one component selected from the group consisting of KCl, bovine serum albumin, and a surfactant; The method.

[27] The method according to

[26] , wherein the surfactant is a cationic surfactant.

[28] The method of

[26] , further comprising heating the sample prior to step (a) or step (b).

[0179] SEQUENCE LISTING <110> QUEST DIAGNOSTICS INVESTMENTS INCORPORATED <120> DIRECT AMPLIFICATION AND DETECTION OF VIRAL AND BACTERIAL PATHOGENS <130> PA24-173 <150> US 61 / 505,055 <151> 2011-07-06 <150> US 61 / 552,405 <151> 2011-10-27 <160> 31 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 1 acgctcaccg tgcccagtga gcgt 24 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 2 ggcattttgg acaaagcgtc ta 22 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 3 tcttgtcacc tctgactaag gggat 25 <210> 4 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <220> <221> modified_base <222> (7)..(7) <223> Inosine <400> 4 ccgcggnatt gcaaaggatg taatggaagt gccgcgg 37 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <220> <221> modified_base <222> (17)..(17) <223> Inosine <400> 5 gagctgaatt tcccatngag ct 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 6 agctgcaaag caacattgga g 21 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 7 acgcgcttca cgaaggctcc acatacacag cgcgt 35 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 8 ttttctagga cattgtaytg aacag 25 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 9 gcaaatatgg aaacatacgt gaacaa 26 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 10 acgcgcttgg ggcgacagtc acgtcgcgcg t 31 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 11 ctcgtcgaca atggcggaa 19 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 12 ttcagcgacc ccgttagc 18 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 13 agcggcccgg gtgcccggcc agccgct 27 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 14 gaggacgagc tggcctttc 19 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 15 acgcgcttcc gggcgttccg cgagcgcgt 29 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 16 gaggacgagc tggcctttc 19 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 17 ggtggtggac aggtcgtaga g 21 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 18 acgcggcttc tgttgtttcg accgcgt 27 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 19 ccccgcttta acacattcca 20 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 20 gcagttgcaa accgggat 18 <210> 21 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 21 aggccacacg gacacccaaa gtagtcggtg gcct 34 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 22 cccctgaatg cggctaatc 19 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 23 caattgtcac cataagcagc ca 22 <210> 24 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 24 aggcagctca ccatcaataa taactgaacc agttgctgcc t 41 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 25 ggttagattt agatgaaaag agatattatt tta 33 <210> 26 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 26 actaatcact aattgagctg tatcagga 28 <210> 27 <211> 122 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 27 ggttagattt agatgaaaag agatattatt ttacagatga atatattgca gcaactggtt 60 cagttattat tgatggtgag gagtattatt ttgatcctga tacagctcaa ttagtgatta 120 gt 122 <210> 28 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 28 agcggcactc caaaaatcag cagctatcaa agcaggtgtg ccgct 45 <210> 29 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 29 aagcttaata tcttctgcgc ttcgt 25 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 30 taacccagta tttgccgatc aa 22 <210> 31 <211> 127 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 31 taacccagta tttgccgatc aaaactttgc tcgtaacgaa aaagaagcaa aagatagcgc 60 tatcacattt atccaaaaat cagcagctat caaagcaggt gcacgaagcg cagaagatat 120 tagctt 127

Claims

1. A reagent mix comprising a primer pair specific to a target nucleic acid derived from a microorganism not extracted from a biological sample, the primer pair being one of the following: A forward primer comprising SEQ ID NOs: 24-25; A forward primer comprising SEQ ID NOs: 28-29; A forward primer comprising SEQ ID NO: 1-2; A forward primer comprising SEQ ID NO: 4-5; A forward primer comprising SEQ ID NOs: 7-8; A forward primer comprising SEQ ID NOs: 10-11; A forward primer comprising SEQ ID NOs: 13-14; A forward primer comprising SEQ ID NOs: 15-16; A forward primer comprising SEQ ID NOs: 18-19; and Forward primer comprising SEQ ID NOs: 21-22 A reagent mix comprising a Scorpion primer selected from the group consisting of:

2. 2. The reagent mix of claim 1, wherein the primer pair comprises a reverse primer selected from the group consisting of SEQ ID NOs: 26, 30, 3, 6, 9, 12, 17, 20 and 23.

3. 2. The reagent mix of claim 1, wherein the Scorpion primer comprises a fluorophore selected from the group consisting of FAM, JOE, CFR 610, Quasar 670, and CAL Fluor Red 610.

4. The reagent mix of claim 1 , wherein the Scorpion primer is labeled with BHQ-1 or BHQ-2.

5. The reagent mix of claim 1 , further comprising a buffer.

6. 6. The reagent mix of claim 5, wherein the buffer comprises albumin.

7. The reagent mix of claim 6, wherein the albumin is BSA.

8. 10. The reagent mix of claim 1, wherein the reagent mix further comprises potassium chloride (KCl).

9. 9. The reagent mix of claim 8, wherein KCl is present at a concentration of 5 mM to 50 mM.

10. The reagent mix of claim 1 , wherein the reagent mix further comprises a surfactant.

11. The reagent mix of claim 10 , wherein the surfactant is a cationic surfactant.

12. The reagent mix of claim 1 , further comprising a DNA polymerase.

13. 13. The reagent mix of claim 12, wherein the DNA polymerase is T. aquaticus (Taq) DNA polymerase.

14. The primer pair is as follows: a forward Scorpion primer comprising SEQ ID NO:24-25, and a reverse primer comprising SEQ ID NO:26; a forward Scorpion primer comprising SEQ ID NO:28-29, and a reverse primer comprising SEQ ID NO:30; a forward Scorpion primer comprising SEQ ID NO:1-2, and a reverse primer comprising SEQ ID NO:3; a forward Scorpion primer comprising SEQ ID NO:4-5, and a reverse primer comprising SEQ ID NO:6; a forward Scorpion primer comprising SEQ ID NO:7-8, and a reverse primer comprising SEQ ID NO:9; a forward Scorpion primer comprising SEQ ID NO:10-11, and a reverse primer comprising SEQ ID NO:12; a forward Scorpion primer comprising SEQ ID NO:13-14, and a reverse primer comprising SEQ ID NO:17; a forward Scorpion primer comprising SEQ ID NO:15-16, and a reverse primer comprising SEQ ID NO:17; a forward Scorpion primer comprising SEQ ID NO:18-19, and a reverse primer comprising SEQ ID NO:20; and A forward Scorpion primer comprising SEQ ID NO:21-22, and a reverse primer comprising SEQ ID NO:

23.

2. The reagent mix of claim 1, selected from the group consisting of:

15. below: a forward Scorpion primer comprising SEQ ID NO:24-25, and a reverse primer comprising SEQ ID NO:26; a forward Scorpion primer comprising SEQ ID NO:1-2, and a reverse primer comprising SEQ ID NO:3; a forward Scorpion primer comprising SEQ ID NO:4-5, and a reverse primer comprising SEQ ID NO:6; a forward Scorpion primer comprising SEQ ID NO:7-8, and a reverse primer comprising SEQ ID NO:9; a forward Scorpion primer comprising SEQ ID NO:10-11, and a reverse primer comprising SEQ ID NO:12; a forward Scorpion primer comprising SEQ ID NO:13-14, and a reverse primer comprising SEQ ID NO:17; a forward Scorpion primer comprising SEQ ID NO:15-16, and a reverse primer comprising SEQ ID NO:17; a forward Scorpion primer comprising SEQ ID NO:18-19, and a reverse primer comprising SEQ ID NO:20; and A forward Scorpion primer comprising SEQ ID NO:21-22, and a reverse primer comprising SEQ ID NO:

23.

2. The reagent mix of claim 1, further comprising a second primer pair selected from the group consisting of:

16. The reagent mix of claim 1 , wherein the biological sample is obtained from a human.

17. 2. The reagent mix of claim 1, wherein the biological sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, oral fluid, and stool.

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