Quantitative PCR with internal control

Abstract

A method for quantifying DNA in a specimen according to the present invention comprises: a step for adding, to a first container containing a specimen and a second container containing a known amount of standard DNA, the same number of copies of internal control DNA, a PCR primer pair for amplifying the DNA in the specimen and the standard DNA, a PCR primer pair for amplifying the internal control DNA, an oligonucleotide fluorescence-labeled probe for detecting the DNA in the specimen and the standard DNA, an oligonucleotide fluorescence-labeled probe for detecting the internal control DNA, and a PCR buffer solution containing a DNA polymerase to perform PCR; a step for comparing Ct values for the internal control DNA contained in the first container and the second container and measuring the difference between the Ct values in the PCR; a step for correcting the Ct value for the DNA in the specimen contained in the first container on the basis of the difference between the Ct values; and a step for measuring the amount of DNA in the specimen from the calibration curve plotted on the basis of the amount of standard DNA contained in the second container and the corrected Ct value. Thus, the target DNA in the specimen can be more accurately quantified.

Description

[Technical Field]

[0001] The present invention relates to a method for quantifying DNA using PCR and a kit for carrying out the method. [Background technology]

[0002] The target DNA in a sample can be amplified and quantified by PCR (polymerase chain reaction). One method for quantifying the target DNA is real-time PCR. In this method, a dilution series of standard DNA with known copy numbers is prepared, and while these are amplified by PCR, the amount of amplified product is detected in real time to create a calibration curve. A calibration curve can be created, for example, by plotting the Ct value of each dilution on a graph with the vertical axis representing the copy number of the standard DNA contained in each dilution in the dilution series and the horizontal axis representing the Ct (threshold cycle) value. The copy number of the target DNA in a sample can be determined by calculating the Ct value of the target DNA and plotting this as the calibration curve. Apply to The dilution series of standard DNA is prepared in a container separate from the container used to prepare the sample. In real-time PCR, these containers are usually wells of a PCR plate or PCR tubes. Summary of the Invention [Problem to be solved by the invention]

[0003] PCR is typically performed using DNA extracted and purified from a sample. However, the process of extracting and purifying DNA from a sample is complicated and time-consuming. On the other hand, in direct PCR, when the target of detection is DNA, the sample is dissolved by chemical treatment or in the heat denaturation step of PCR to liberate the DNA. In direct PCR, when the target of detection is RNA, the RNA is liberated by chemical treatment or heat treatment of the sample, and the complementary strand DNA converted from the RNA by reverse transcription is directly amplified from the sample. Therefore, direct PCR is easy to operate and requires less time.

[0004] However, when quantifying target DNA in a sample using a calibration curve by direct PCR, the PCR reaction solution containing the sample contains sample-derived components, such as PCR inhibitors, while the PCR reaction solution containing the standard DNA used to create the calibration curve does not contain such sample-derived components, resulting in PCR being performed under conditions with variations in PCR reaction solution composition. Furthermore, the degree of influence of PCR inhibitors on PCR varies from sample to sample, which can lead to differences in PCR progress between wells. Therefore, while the signal values ​​obtained from real-time PCR may be affected by differences in PCR reaction solution composition and / or sample-to-sample variations in the PCR process, there has been no way to confirm whether the amount of target DNA present in the sample is accurately measured based on the calibration curve.

[0005] The object of the present invention is to provide a method for more accurately quantifying target DNA in a sample, and a kit for carrying out the method, by measuring the effect on the signal value obtained from real-time PCR of differences in the composition of the reaction solution between a PCR reaction solution containing standard DNA for preparing a calibration curve and a PCR reaction solution containing target DNA in a sample and / or differences between samples in the PCR process, etc., and detecting errors in the signal value and correcting the detected errors. [Means for solving the problem]

[0006] That is, the object of the present invention is achieved by the following inventions. [1] A method for quantifying DNA in a sample, comprising: adding the same copy number of internal control DNA, a PCR primer pair for amplifying the DNA in the sample and the standard DNA, a PCR primer pair for amplifying the internal control DNA, fluorescently labeled oligonucleotide probes for detecting the DNA in the sample and the standard DNA, fluorescently labeled oligonucleotide probes for detecting the internal control DNA, and a PCR buffer containing DNA polymerase to a first container containing a sample and a second container containing a known amount of standard DNA, respectively, and performing PCR; comparing the Ct values ​​for the internal control DNA contained in the first container and the second container in the PCR and measuring the difference between the Ct values; correcting the Ct value for the DNA in the sample contained in the first container based on the difference between the Ct values; and measuring the amount of DNA in the sample from a calibration curve prepared based on the amount of standard DNA contained in the second container and the corrected Ct value; A quantitative method comprising:

[0007] [2] The method according to [1], wherein the specimen is a sample selected from the group consisting of a biological sample, a biologically derived sample, an environmental sample, and an environmentally derived sample. [3] The method according to [1], wherein the specimen is a sample selected from the group consisting of a fecal sample, a fecal-derived sample, a vomit sample, and a vomit-derived sample. [4] The method according to any one of [1] to [3], wherein the specimen contains a pathogen. [5] The method according to [4], wherein the pathogen is a virus, bacterium, fungus or protozoan. [6] The method according to any one of [1] to [5], wherein the internal control DNA has a chain length of 50 to 200 bp and a GC content of 40 to 60%. [7] The method according to any one of [1] to [6], wherein the PCR buffer solution contains a surfactant. [8] The method according to [7], wherein the surfactant is a nonionic surfactant. [9] The method according to any one of [1] to [8], wherein the PCR buffer is a Tris buffer containing KCl, MgCl2, and a dNTP mix (a mixture consisting of dATP, dGTP, dCTP, and dTTP).

[10] The method according to any one of [1] to [9], wherein the PCR buffer solution contains a negatively charged substance of biological origin that adsorbs to DNA polymerase and a positively charged substance of biological origin that adsorbs to DNA, which bind to a substance that inhibits PCR, and neutralize the PCR-inhibiting action of the negatively charged substance and the positively charged substance.

[0008]

[11] A kit for quantifying DNA in a sample, comprising: internal control DNA; PCR primer pairs for amplifying the DNA in the sample and the standard DNA; a PCR primer pair for amplifying the internal control DNA; an oligonucleotide fluorescently labeled probe for detecting DNA in the sample and the standard DNA; an oligonucleotide fluorescently labeled probe for detecting the internal control DNA; DNA polymerase; and PCR buffer; Kit including:

[0009]

[12] The kit according to

[11] , wherein the internal control DNA has a chain length of 50 to 200 bp and a GC content of 40 to 60%.

[13] The kit according to

[11] or

[12] , wherein the PCR buffer solution contains a surfactant.

[14] The kit according to

[13] , wherein the surfactant is a nonionic surfactant.

[15] The kit according to any one of

[11] to

[14] , wherein the PCR buffer is a Tris buffer containing KCl, MgCl2, and a dNTP mix (a mixture consisting of dATP, dGTP, dCTP, and dTTP).

[16] The kit according to any one of

[11] to

[15] , wherein the PCR buffer solution contains a negatively charged substance of biological origin that adsorbs to DNA polymerase and a positively charged substance of biological origin that adsorbs to DNA, which binds to a substance that inhibits PCR, and neutralizes the PCR-inhibiting action of the negatively charged substance and the positively charged substance. [Effects of the Invention]

[0010] Since real-time PCR is performed by adding the same copy number of internal control DNA to a first container containing a sample and a second container containing a known amount of DNA for creating a standard curve, even if positive or negative errors occur in the signal values ​​obtained from real-time PCR due to differences in the composition of the reaction solution between the PCR reaction solution for the sample and the PCR reaction solution for creating the standard curve and / or differences between samples in the PCR process, such errors can be detected by comparing the amplification curves for the internal control DNA contained in the first container and the second container.Furthermore, the amount of target DNA in the sample is measured based on the detected errors, allowing for more accurate quantification of the target DNA. DETAILED DESCRIPTION OF THE INVENTION

[0011] [Specimen] Specimens in the present invention include biological samples, biologically derived samples, environmental samples, and environmentally derived samples. Biological samples include cell, tissue, and / or organ homogenates and extracts. Tissues or organs include the brain, spinal cord, bone marrow, conjunctiva, cornea, vitreous body, heart, mitral valve, tricuspid valve, lung, pleura, liver, spleen, peritoneum, intestine, lymph nodes, and skin. Biological samples also include blood and blood-related samples, including whole blood, plasma, and serum, lymph, saliva, nasal secretions, throat swabs, nasal swabs, sweat, tears, tissue fluids (intertissue fluid, intercellular fluid, and interstitial fluid), body cavity fluids (ascites, pleural effusion, pericardial fluid, cerebrospinal fluid, synovial fluid, and aqueous humor), and exudates (such as pleural effusion or ascites) from the thoracic, peritoneal, cranial, or spinal canal. Biological samples may be centrifuged, and the supernatant or sediment obtained by centrifugation may be used as the specimen. Biological samples also include those obtained by mixing the biological sample with a culture medium, a buffer solution, a specimen preservation solution, etc. Examples of the buffer solution include, but are not limited to, phosphate buffer, Tris buffer, borate buffer, and Good's buffer such as HEPES. Biologically derived samples include those obtained by treating the biological sample, for example, by sonication. Environmental samples include all samples including air, soil, dust, water, etc. Environmentally derived samples include those obtained by treating the environmental sample, for example, by sonication.

[0012] In another embodiment of the present invention, the specimen may include a fecal sample, a fecal-derived sample, a vomit sample, and a vomit-derived sample. Fecal and vomit samples can be used as specimens directly, or may be diluted or suspended in distilled water, physiological saline, or a buffer solution. Examples of the buffer solution include, but are not limited to, phosphate buffer, Tris buffer, borate buffer, and Good's buffer such as HEPES. The suspension of the sample may be centrifuged, and the supernatant may be used as the specimen. Fecal and vomit-derived specimens include swab samples. Swab samples are obtained by wiping hands, tableware, kitchen equipment, toilet equipment, or household equipment with a cotton swab or cotton wool, and then dissolving the wiped material in a phosphate buffer solution or the like.

[0013] The specimen of the present invention may contain a pathogen. Examples of pathogens include viruses, bacteria, fungi, and protozoa. These viruses include DNA and RNA viruses. DNA viruses are viruses that have DNA as their genome, including, but not limited to, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus type 6 (HHV-6), adenovirus, and papillomavirus. RNA viruses are viruses that have RNA as their genome, including, but not limited to, enveloped viruses with a lipid bilayer membrane, such as coronavirus, human immunodeficiency virus, hepatitis C virus, Japanese encephalitis virus, and dengue virus, as well as non-enveloped viruses, such as norovirus, rotavirus, and rhinovirus. The genomic RNA of an RNA virus can be converted into "DNA in a specimen" by generating cDNA through reverse transcription. Examples of bacteria include, but are not limited to, Staphylococcus aureus, Chlamydia, Salmonella, Bacillus cereus, Vibrio parahaemolyticus, enterohemorrhagic Escherichia coli O157, and Treponema pallidum, while examples of protozoa include Toxoplasma gondii and Entamoeba histolytica. DNA in a sample can be amplified and detected by PCR by selecting the right primer pair (forward and reverse) to amplify the target gene region.

[0014] There are no particular restrictions on the standard DNA used in the present invention, as long as it has the sequence of the target DNA in the sample. However, it is preferable that the standard DNA be similar in shape and size to the target DNA in order to match the PCR amplification efficiency as closely as possible.

[0015] [Real-time PCR] (container) The first and second containers are preferably wells of a PCR plate or PCR tubes. PCR plates of, for example, 96-well and 386-well formats can be used. PCR tubes can be single tubes or 2- to 12-strip tubes with a volume size of about 0.1 to 0.5 mL. Polypropylene or the like can be used as the material for the first and second containers. The tube color is preferably uncolored or white.

[0016] (PCR buffer) The PCR buffer contains KCl, MgCl, and a dNTP mix (a mixture of deoxyribonucleotide 5'-triphosphate; dATP, dGTP, dCTP, and dTTP). The PCR buffer is preferably, but not limited to, a Tris-HCl buffer. Those skilled in the art can determine appropriate concentrations of dNTP, MgCl, KCl, and the buffer. For example, the buffer may contain 1.5 mM MgCl, 35 mM KCl, 200 μM each of dNTP, and 10 mM Tris-HCl. In one embodiment of the present invention, the PCR buffer contains a biologically derived negatively charged substance (e.g., certain sugars and dyes) that adsorbs to DNA polymerase and a biologically derived positively charged substance (e.g., certain proteins) that adsorbs to DNA and binds to PCR-inhibiting substances, neutralizing the PCR-inhibiting effects of the negatively and positively charged substances. Ampdirect Plus (registered trademark, Shimadzu Corporation), a gene amplification reagent, can be used as the PCR buffer.

[0017] The PCR buffer solution contains an internal control DNA, and the same number of copies of the internal control DNA are added to the first and second containers, respectively. The PCR buffer solution further contains a PCR primer pair for amplifying the DNA in the sample and the standard DNA, a PCR primer pair for amplifying the internal control DNA, fluorescently labeled oligonucleotide probes for detecting the amplified DNA in the sample and the standard DNA, a fluorescently labeled oligonucleotide probe for detecting the amplified internal control DNA, and a DNA polymerase.

[0018] If the specimen contains RNA, the PCR buffer solution may further contain a reverse transcriptase and a reverse transcription primer to generate cDNA by reverse transcription. The reverse transcriptase is an enzyme that generates single-stranded complementary DNA (cDNA) using RNA as a template. The reverse transcriptase is not particularly limited as long as it catalyzes the reverse transcription reaction. Examples of suitable reverse transcriptases include RNA-dependent DNA polymerases derived from RNA viruses such as avian myeloblastosis virus (AMV), Moloney murine leukemia virus (M-MLV), and human immunodeficiency virus (HIV), as well as their mutants. The reverse transcription primer may be a primer that specifically hybridizes to the target RNA sequence, an oligo(dT) primer, or a random primer.

[0019] (internal control DNA) The internal control DNA has a sequence that does not hybridize with PCR primers for amplifying the DNA in the sample and the standard DNA, or with probes for detecting the amplified DNA in the sample and the standard DNA. Therefore, the internal control DNA is amplified and detected independently of the DNA in the sample and the standard DNA. For this reason, the sequence of the internal control DNA is modified according to the sequence of the DNA to be amplified in the sample, and is preferably an artificially synthesized sequence that does not exist in nature. The chain length of the internal control DNA is preferably approximately 50 to 200 bp, more preferably 80 to 120 bp, to improve amplification efficiency. Furthermore, the GC content of the internal control DNA is preferably approximately 40 to 60% to avoid a decrease in amplification efficiency. The term "approximately" in the chain length and GC content refers to the chain length being outside the range of 50 to 200 bp and the GC content being outside the range of 40 to 60%, as long as the amplification efficiency is not decreased.

[0020] (PCR primer pair for DNA amplification) The PCR primer pair for amplifying the sample DNA and the standard DNA is an oligonucleotide (forward and reverse) that hybridizes to the target base sequence under stringent conditions. The PCR primer pair for amplifying the internal control DNA is an oligonucleotide (forward and reverse) that hybridizes to a predetermined base sequence of the internal control DNA under stringent conditions. These stringent conditions refer to conditions under which the binding between the template DNA and the primers is specific during annealing in PCR, the step in which the primers bind to the template DNA. The PCR primer pair for amplifying the sample DNA and the standard DNA does not hybridize to the internal control DNA, and the PCR primer pair for amplifying the internal control DNA does not hybridize to the sample DNA or the standard DNA. These primers preferably have a base length of 15 to 30 bases. The base sequences of the primers used must be designed so that amplification of the target gene region by each PCR primer pair proceeds smoothly in a single PCR reaction system.

[0021] (Fluorescently labeled oligonucleotide probe) In the present invention, PCR products can be detected by real-time measurement. Real-time measurement of PCR products is also called real-time PCR. In the present invention, fluorescently labeled oligonucleotide probes are used to detect PCR products by fluorescence. The fluorescently labeled oligonucleotide probes for detecting the sample DNA and standard DNA hybridize under stringent conditions to PCR products amplified with PCR primer pairs for amplifying the sample DNA and standard DNA, but do not hybridize to PCR products amplified with PCR primer pairs for amplifying the internal control DNA. The fluorescently labeled oligonucleotide probe for detecting the internal control DNA hybridizes under stringent conditions to PCR products amplified with PCR primer pairs for amplifying the internal control DNA, but does not hybridize to PCR products amplified with PCR primer pairs for amplifying the sample DNA and standard DNA. These stringent conditions refer to conditions under which specific hybrids are formed, but nonspecific hybrids are not formed, between the nucleic acid fragments amplified by PCR and the fluorescently labeled oligonucleotide probes upon annealing. The fluorescently labeled oligonucleotide probe used in the present invention preferably has a base length of 15 to 25 bases.

[0022] Examples of the fluorescently labeled probe include, but are not limited to, hydrolysis probes, molecular beacons, and cycling probes. Hydrolysis probes are oligonucleotides modified with a fluorescent dye at the 5' end and a quencher at the 3' end. During PCR annealing, hydrolysis probes specifically hybridize to template DNA, but the presence of a quencher on the probe suppresses fluorescence emission even when irradiated with excitation light. In the subsequent extension reaction step, the 5'->3' exonuclease activity of Taq DNA polymerase degrades the hydrolysis probe hybridized to the template DNA, liberating the fluorescent dye from the probe. This releases the quencher-mediated suppression of fluorescence emission, allowing the probe to emit fluorescence. The amount of amplified product produced can be measured by measuring this fluorescence intensity. Examples of the fluorescent dyes include, but are not limited to, FAM (6-carboxyfluorescein), ROX (6-carboxy-X-rhodamine), Cy3 and Cy5 (cyanine dyes), and HEX (4,7,2',4',5',7'-hexachloro-6-carboxyfluorescein). Examples of the quenchers include, but are not limited to, TAMRA (registered trademark), BHQ (Black Hole Quencher, registered trademark) 1, BHQ2, MGB-Eclipse (registered trademark), and DABCYL.

[0023] The fluorescently labeled oligonucleotide probes for detecting the DNA and standard DNA in the sample and the fluorescently labeled oligonucleotide probe for detecting the internal control DNA are bound to different fluorescent dyes. Because the fluorescent dyes bound to each probe are different, PCR products from the PCR primer pairs for amplifying the DNA in the sample and the standard DNA and the PCR primer pair for amplifying the internal control DNA can be measured separately. The combination of different fluorescent dyes is not particularly limited, as long as they have different fluorescent properties and do not interfere with each other in fluorescence measurement.

[0024] (DNA polymerase) The DNA polymerase is a thermostable DNA polymerase derived from thermophilic bacteria, and examples include, but are not limited to, Taq, Tth, KOD, Pfu, and their mutants. To avoid nonspecific amplification by the DNA polymerase, a hot-start DNA polymerase may be used. Examples of hot-start DNA polymerases include BIOTAQ® hot-start DNA polymerase. Hot-start DNA polymerases include DNA polymerases bound to anti-DNA polymerase antibodies and DNA polymerases with thermosensitive chemical modifications at the enzyme active site. Both of these can be used in the present invention.

[0025] [Preparation of PCR reaction solution] A PCR reaction solution for the sample is prepared by adding to a first container a sample, an internal control DNA, a PCR primer pair for amplifying DNA in the sample, a PCR primer pair for amplifying the internal control DNA, a fluorescently labeled oligonucleotide probe for detecting DNA in the sample, a fluorescently labeled oligonucleotide probe for detecting the internal control DNA, and a PCR buffer solution containing DNA polymerase.

[0026] The sample added to the first container is preferably mixed with the PCR buffer containing a surfactant to liberate DNA or RNA contained in the sample into the PCR reaction solution. The surfactant contained in the PCR buffer can be an anionic surfactant, cationic surfactant, amphoteric surfactant, or nonionic surfactant, but a nonionic surfactant is preferred. Nonionic surfactants include, but are not limited to, Tween® 20 (polyoxyethylene sorbitan monolaurate), Tween 80 (polyoxyethylene sorbitan monooleate), Triton® X-100 (polyethylene glycol mono-4-octylphenyl ether), Nonidet® P-40 (octylphenyl-polyethylene glycol, NP-40), and Brij®-35 (polyoxyethylene lauryl ether). The concentration of the surfactant used is preferably 0.05 to 5% (w / v) when mixed with the sample. The PCR buffer may further contain proteinase K. Proteinase K has the effect of inactivating DNA and RNA degrading enzymes, and is preferably present at 100 to 300 μg / mL when mixed with the specimen.

[0027] To liberate RNA from a sample containing an RNA virus such as coronavirus, the sample may be mixed with a sample treatment solution containing sodium hydroxide as the main component and incubated at room temperature to 95°C, preferably 80 to 95°C, for 3 to 5 minutes. Room temperature is typically around 25°C. In addition to sodium hydroxide, the sample treatment solution may contain a metal chelating agent such as glycol ether diamine tetraacetic acid and / or a reducing agent such as dithiothreitol to efficiently perform RT (reverse transcription)-PCR and improve measurement accuracy. The sample incubated with the sample treatment solution can be mixed with the PCR buffer solution and subjected to RT-PCR.

[0028] A PCR reaction solution for preparing a calibration curve is prepared by adding standard DNA, internal control DNA, a PCR primer pair for amplifying the standard DNA, a PCR primer pair for amplifying the internal control DNA, a fluorescently labeled oligonucleotide probe for detecting the standard DNA, a fluorescently labeled oligonucleotide probe for detecting the internal control DNA, and a PCR buffer containing DNA polymerase to a second container. The internal control DNA added to the second container is a PCR reaction solution for preparing a calibration curve. DNA The PCR primer pair for amplifying the DNA in the sample added to the first container and the PCR primer pair for amplifying the standard DNA added to the second container are preferably the same, but may be different. Similarly, the fluorescently labeled oligonucleotide probe for detecting the DNA in the sample added to the first container and the fluorescently labeled oligonucleotide probe for detecting the standard DNA added to the second container are preferably the same, but may be different.

[0029] Multiple second containers are prepared, each containing a known amount of serially diluted standard DNA. These serially diluted standard DNAs are used to generate a calibration curve. The second containers are subjected to the same procedures as the first container, except that the standard DNA is added instead of the sample.

[0030] [Measurement of Ct value] In real-time PCR product measurement, the amplification curve of the PCR product is monitored using a fluorescence filter corresponding to the fluorescent dye used. The amplification curve can be created by plotting fluorescence intensity versus PCR cycle number. The Ct value (Threshold Cycle) is calculated, for example, as the PCR cycle number corresponding to the point where the amplification curve intersects with a threshold set in the rising region of the amplification curve. Real-time PCR is performed on a dilution series of a known amount of standard DNA contained in a second container, and the Ct value corresponding to the initial concentration of the standard DNA at each point in the dilution series is calculated, allowing a calibration curve for quantifying DNA in a sample to be obtained. The calibration curve plot is usually created with the Ct value of the standard DNA on the horizontal axis and the initial concentration of the standard DNA on the vertical axis. DNA in a sample can be quantified by applying the Ct value obtained for the sample to the calibration curve.

[0031] Because the same copy number of the internal control DNA is added to the first container containing the sample and the second container containing the standard DNA, the amount of PCR product produced by the internal control DNA is theoretically the same in each container, resulting in the same Ct value. In this case, there is no difference in the signal values ​​obtained from the real-time PCR method for each container. However, if positive or negative errors occur in the signal values ​​obtained from the real-time PCR method due to differences in the composition of the reaction solution between the PCR reaction solution for the sample and the PCR reaction solution used to create the standard curve and / or differences between containers (wells / tubes) in the PCR process, it becomes difficult to accurately quantify the DNA in the sample. This error is the difference in the Ct values ​​of the internal control DNA in the first container (for the sample) and the second container (for the standard curve). Therefore, the Ct value for the DNA in the sample contained in the first container can be corrected based on this difference. By applying this corrected Ct value to the standard curve, the DNA in the sample can be more accurately quantified.

[0032] For example, the corrected value (Ctcv) of the Ct value (Ct) for DNA in the sample contained in the first container can be calculated by the following formula. Ct value for the internal control DNA contained in the first container (for specimen); Ct1 Ct value for the internal control DNA contained in the second container (for the calibration curve); Ct2 Ctcv=Ct2 / Ct1×Ct Ct1, Ct2 and Ct may be average values ​​obtained from multiple containers.

[0033] 〔kit〕 In the kit of the present invention, the internal control DNA, PCR primer pairs for amplifying the DNA in the sample and the standard DNA, PCR primer pairs for amplifying the internal control DNA, fluorescently labeled oligonucleotide probe, DNA polymerase, and PCR buffer may each be contained independently in different containers. For ease of operation, the internal control DNA, PCR primer pairs for amplifying the DNA in the sample and the standard DNA, PCR primer pairs for amplifying the internal control DNA, fluorescently labeled oligonucleotide probe, DNA polymerase, and PCR buffer may each be mixed in predetermined amounts and contained in a single container. Furthermore, depending on the intended use of the kit, the kit components may be distributed and contained in two to four containers.

Claims

1. A method for quantifying DNA in a sample, comprising: adding the same copy number of internal control DNA, a PCR primer pair for amplifying the DNA in the sample and the standard DNA, a PCR primer pair for amplifying the internal control DNA, fluorescently labeled oligonucleotide probes for detecting the DNA in the sample and the standard DNA, fluorescently labeled oligonucleotide probes for detecting the internal control DNA, and a PCR buffer containing DNA polymerase to a first container containing a sample and a second container containing a known amount of standard DNA, respectively, and performing PCR; a step of comparing the Ct values ​​for the internal control DNA contained in the first container and the second container in the PCR, and calculating a ratio between the two Ct values; correcting the Ct value for the DNA in the sample contained in the first container based on the ratio of the Ct values; creating a calibration curve based on the known amount of the standard DNA contained in the second container and the Ct value for the standard DNA in the PCR; and measuring the amount of DNA in the sample from the calibration curve and the corrected Ct value; A quantitative method comprising:

2. 2. The method of claim 1, wherein the specimen is a sample selected from the group consisting of a biological sample, a biologically derived sample, an environmental sample, and an environmentally derived sample.

3. 2. The method of claim 1, wherein the specimen is a sample selected from the group consisting of a fecal sample, a fecal-derived sample, a vomit sample, and a vomit-derived sample.

4. The method of any one of claims 1 to 3, wherein the specimen comprises a pathogen.

5. 5. The method of claim 4, wherein the pathogen is a virus, bacterium, fungus, or protozoan.

6. The method according to any one of claims 1 to 3, wherein the internal control DNA has a chain length of 50 to 200 bp and a GC content of 40 to 60%.

7. The method according to any one of claims 1 to 3, wherein the PCR buffer comprises a surfactant.

8. The method of claim 7 , wherein the surfactant is a non-ionic surfactant.

9. The PCR buffer contains KCl, MgCl ２ and a dNTP mix (a mixture of dATP, dGTP, dCTP, and dTTP).

10. The method according to any one of claims 1 to 3, wherein the PCR buffer solution contains a negatively charged substance of biological origin that adsorbs to DNA polymerase and a positively charged substance of biological origin that adsorbs to DNA, which binds to a substance that inhibits PCR, and neutralizes the PCR-inhibiting action of the negatively charged substance and the positively charged substance.