WO2022059555A1 - Improved multiplex pcr testing method - Google Patents

Abstract

The purpose of the present invention is to provide an improved multiplex PCR testing method that has excellent workability and that, while simple, can detect a plurality of target nucleic acids with high sensitivity. The present invention provides a method for using one PCR reaction solution to test for the presence/absence of two or more target nucleic acids in a sample, said method being characterized by comprising the following steps: (1) a step for preparing a sample mixed solution that includes a visible pigment and a sample that has not undergone a nucleic acid isolation process; (2) a step for mixing the sample mixed solution prepared in step (1) and the PCR reaction solution, which contains a DNA polymerase that is resistant to foreign substances; (3) a step for performing a PCR reaction after sealing a reaction container; and (4) a step for using two or more types of fluorescent compounds to detect the two or more target nucleic acids.

Description

Improved multiplex PCR test method

The present invention relates to a method for detecting nucleic acid derived from a pathogenic microorganism such as a virus by nucleic acid amplification. More specifically, without isolating and purifying the nucleic acid in advance from the sample, a sample mixture prepared by mixing the sample with a visible dye to prepare a colored sample mixture is prepared, and a reaction of a real-time polymerase chain reaction or a real-time reverse transcription polymerase chain reaction is performed. It relates to the detection of nucleic acid derived from a pathogenic microorganism by adding a liquid. The method of the present invention can detect pathogenic microorganism-derived nucleic acids in, for example, pharyngeal and nasal swab samples, saliva and sputum samples, fecal samples, blood samples, mouthwash, tears, and environmental wiping samples. be. The present invention can also be used for life science research, clinical diagnosis, food hygiene inspection, environmental inspection and the like.

Nucleic acid amplification method is a technology that amplifies several copies of target nucleic acid to a level that can be visualized, that is, to hundreds of millions of copies or more. It is also widely used in microbiological examinations and the like. A typical nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR includes (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) the primer using a DNA polymerase. This is a method in which the target nucleic acid in the sample is amplified by repeating the three steps of elongation as one cycle. Annealing and elongation may be performed at the same temperature in two steps.

When analyzing RNA, reverse transcription (RT), which converts the template RNA into cDNA, is performed as a pre-stage of this PCR. This is called RT-PCR. This RT-PCR is a one-enzyme system one-step RT in which (1) RT and PCR are carried out discontinuously, two-step RT-PCR, and (2) RT and PCR are carried out continuously using a single enzyme. -PCR, (3) Using two types of enzymes, reverse transcriptase and DNA polymerase, RT and PCR are roughly divided into three types: two-enzyme system 1-step RT-PCR.

PCR and RT-PCR are widely used for genetic testing and pathogenic microbiological testing. Coronavirus, which is one of the pathogenic RNA viruses, is a typical example of virus testing. Coronavirus is a causative virus that causes respiratory infections including colds, and it is said that about 10 to 35% of coronaviruses are caused by the coronavirus during the cold season. It is also known that mutant viruses occur, and rarely SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, and new coronavirus infection (COVID-19) coronavirus (SARS). It is known that those that cause fatal and serious respiratory diseases such as -nCOV-2 or SARS-CoV-2) occur.

There are many pathogenic microorganisms that cause respiratory diseases, but it is difficult to identify the cause from the symptoms of the disease. In addition, since the treatment method differs depending on the pathogenic microorganism that causes the disease, early identification of the cause is very important in terms of reducing the physical burden on the patient's treatment and the mortality rate. Therefore, it goes without saying that it is important in clinical diagnosis, food hygiene inspection, environmental inspection, etc. to detect and identify pathogenic microorganisms that cause respiratory infections such as coronavirus easily, quickly, and with high sensitivity. ..

By the way, the work of dispensing a sample containing a sample or a PCR reaction solution into a reaction vessel such as a PCR tube or a PCR plate can be performed hundreds or thousands of times depending on the number of samples. In the continuous dispensing and addition work into the reaction vessel, mistakes such as omission of dispensing into the reaction vessel and multiple dispensing may occur. In particular, if a dispensing error occurs when a sample including a sample is added, the test cannot be performed correctly, and further work is required due to retesting or the like, resulting in time and financial loss.

Generally, since the amount of sample added to PCR is very small, it is difficult to determine whether or not the sample has been dispensed into the reaction vessel and the amount of the dispensed sample. This point can be a problem because it is easy to overlook, especially when inspecting a large number of samples. In addition, in a coronavirus test using saliva or nasopharyngeal swab sample as a sample, the sample may be translucent and have low turbidity, so it may be more difficult to judge the amount to be dispensed. In recent years, PCR reaction solutions and additive buffers containing visible dyes have been developed to improve visibility during dispensing, and when a method is used to easily confirm the presence or absence of dispensing and the amount of dispensing after dispensing. There is. However, in the method for detecting an amplification product using a fluorescent compound, it is often confirmed that the detected fluorescence intensity is lowered depending on the combination of the color tone of the visible dye and the excitation wavelength and the fluorescence wavelength of the fluorescent compound.

In addition, when using a sample for which nucleic acid has not been isolated, for example, in a coronavirus test, a PCR reaction inhibitor such as a polysaccharide contained in saliva or nasopharyngeal swab sample by omitting RNA extraction. And insoluble substances are brought in. The presence of such an inhibitor or insoluble substance may cause a decrease in the intensity such as the detected fluorescence intensity in the detection using the fluorescent compound, and a decrease in the detection sensitivity associated therewith.

When performing multiplex PCR in which two or more target nucleic acids are detected in one reaction solution, usually two or more kinds of fluorescent compounds are used. However, in such a situation where a plurality of fluorescent compounds are used and it is necessary to distinguish and detect them, the combination of the excitation wavelength and the fluorescence wavelength becomes more complicated, and the decrease in the fluorescence intensity may become a further problem.

Therefore, there is a demand for the development of a method capable of detecting the presence or absence of a plurality of target nucleic acids with high sensitivity by a simple method while having excellent workability.

WO 2019/212023 Pamphlet Japanese Unexamined Patent Publication No. 2012-2439

Food Hygiene Magazine, Vol. 46, 2005, pp. 235-245 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2005, p. 5452-5456 PublicOnline January 29,2020, https: // doi. org / 10.1016 / S0140-6736 (20) 30251-8 World Health Organization (WHO) homepage (Diagnosis detection of Wuhan coronavirus 2019 by real-timeRT-PCR) National Institute of Infectious Diseases Homepage "Pathogen Detection Manual 2019-nCoV" (https://www.niid.go.jp/niid/images/lab-manual/2019-nCoV20200217.pdf)

An object of the present invention is to use a simple method while having excellent workability when using a sample containing a sample (so-called crude sample) that may contain a contaminating substance or an insoluble substance that inhibits a nucleic acid amplification reaction or detection of a nucleic acid amplification product. It is to provide a method capable of detecting the presence or absence of a plurality of target nucleic acids with high sensitivity.

In view of the above circumstances, the present inventors have conducted diligent research, and as a result, a sample that has not been isolated from nucleic acid and may contain contaminating substances and / or insoluble substances was mixed with a visible dye and colored. After preparing a sample mixture, it is mixed with a PCR reaction solution containing a specific DNA polymerase and subjected to a PCR reaction or an RT-PCR reaction to derive from two or more target nucleic acids (for example, pathogenic microorganisms such as viruses and bacteria). It has been found that it is possible to detect the target nucleic acid).

Conventionally, it has been considered that the inhibition of the PCR reaction and the presence of the visible dye due to the introduction of contaminants into the PCR reaction solution significantly reduce the fluorescence intensity at the time of measurement with a real-time PCR machine and cause a significant decrease in sensitivity. However, quite unexpectedly, if rTth DNA polymerase and Hawk Z05 DNA polymerase, which are heat-resistant DNA polymerases having contamination resistance, and their variants, etc. are used, due to their high reactivity, not only contaminants but also impurities. By obtaining a strong fluorescence intensity that exceeds the effect of the decrease in fluorescence intensity caused by the presence of the visible dye, the decrease in detection sensitivity can be overcome, and two or more target nucleic acids due to two or more types of fluorescent compounds can be obtained. We have found that detection is also possible. As a result, without performing the work of isolating and purifying the nucleic acid from the sample which may contain contaminants in advance, the sample is mixed with the visible dye to prepare a colored sample mixture, and then the PCR reaction solution or the RT-PCR reaction is performed. We have found that nucleic acids derived from a plurality of pathogenic microorganisms can be detected only by adding them to a solution and performing a multiplex RT-PCR reaction, and have completed the present invention.

Typical inventions of the present application are as follows.

[Item 1] A method for inspecting the presence or absence of two or more target nucleic acids in a sample with one PCR reaction solution, which comprises the following steps:

(1) A step of preparing a sample mixture containing a sample that has not been isolated from nucleic acid and a visible dye.

(2) A step of mixing the sample mixture prepared in the above step (1) with a PCR reaction solution containing a DNA polymerase having contamination resistance.

(3) A step of carrying out a PCR reaction after sealing the reaction vessel; and

(4) A step of detecting two or more target nucleic acids with two or more types of fluorescent compounds.

[Item 2] The method according to Item 1, wherein the DNA polymerase having contamination resistance is a DNA polymerase having reverse transcription activity.

[Item 3] The method according to Item 1, wherein the PCR reaction solution further contains reverse transcriptase.

[Item 4] Item 1 to item 1 to which the sample is at least one selected from the group consisting of saliva sample, sputum sample, mouthwash, tears, pharyngeal swab sample, nasal swab sample, fecal sample and wiping test sample. The method according to any one of 3.

[Item 5] The sample is a suspension suspended in at least one selected from the group consisting of water, physiological saline, a buffer solution, and a sputazyme enzyme solution, or a centrifugal supernatant or a concentrate thereof. Item 8. The method according to any one of Items 1 to 4.

[Item 6] The method according to any one of Items 1 to 5, wherein the DNA polymerase having contamination resistance is a DNA polymerase belonging to Family A.

Item 7 The DNA polymerase having contamination resistance is a DNA polymerase having at least one contaminant resistance selected from the group consisting of Tth, Howk Z05 and variants thereof. The method according to any one of 6.

[Item 8] A DNA in which the mutant comprises an amino acid sequence exhibiting 90% or more identity with the amino acid sequence of Tth polymerase (SEQ ID NO: 25) or Hawk Z05 polymerase (SEQ ID NO: 26) and having contamination resistance. Item 6. The method according to Item 7, which exhibits polymerase activity.

[Item 9] The variant comprises an amino acid sequence having one or several amino acid deletions, substitutions and / or additions in the amino acid sequence of Tth polymerase (SEQ ID NO: 25) or Hawk Z05 polymerase (SEQ ID NO: 26). Item 7. The method according to Item 7 or 8, which exhibits DNA polymerase activity having contamination resistance.

[Item 10] The reverse transcriptase is a reverse transcriptase derived from at least one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and variants thereof. The method according to any one of 3 to 9.

[Item 11] The visible dye is purple (400-420 nm), indigo color (420-440 nm), blue (440-490 nm), green (490-570 nm), yellow (570-585 nm), orange (585-620 nm). The method according to any one of Items 1 to 10, wherein the dye exhibits a color tone (maximum absorption wavelength in parentheses) selected from the group consisting of red (620-780 nm), white, black, and gray.

[Item 12] Visible pigments are amaranth, erythrosin, alla red, ponceau, phenol red, orange G, ponso S, cresol red, rose bengal, tartrazine, sunset yellow, fast green, bromocresol green, brilliant blue, indigo. Item 6. The method according to any one of Items 1 to 11, which is at least one selected from the group consisting of carmine, patent blue, bromophenol blue, and bromocresol purp.

[Item 13] As the visible dye, a visible dye-containing solution in which the visible dye is suspended in at least one solvent selected from the group consisting of water, physiological saline, a buffer solution, an acidic solution, an alkaline solution, and an organic solvent. Item 6. The method according to any one of Items 1 to 12.

[Item 14] The method according to any one of Items 1 to 13, wherein the concentration of the visible dye in the visible dye-containing solution is 0.00001% or more.

[Item 15] The method according to any one of Items 1 to 14, wherein the concentration of the visible dye in the PCR reaction solution is 0.000001% or more.

Item 6. The method according to any one of Items 1 to 15, wherein at least one of the fluorescent compounds is a double-stranded DNA-bound fluorescent compound.

[Item 17] Double-stranded DNA-bound fluorescent compounds are SYBR® Green I, SYBR® Gold, SYTO-9, SYTP-13, SYTO-82, EvaGreen®, LCGreen, Item 16. The method according to Item 16, wherein the method is one or more selected from the group consisting of Light Cycler (registered trademark) 480 ResoLight.

[Item 18] The method according to any one of Items 1 to 17, wherein at least one of the fluorescent compounds is a hybridization probe.

Item 19. The method according to Item 18, wherein the hybridization probe is a TaqMan probe.

Item 20. The method according to any one of Items 1 to 19, wherein the two or more kinds of fluorescent compounds include a combination of a hybridization probe and a double-stranded DNA-bound fluorescent compound.

[Item 21] The method according to any one of Items 1 to 20, wherein at least one of the two or more target nucleic acids in the sample is a nucleic acid derived from a pathogenic microorganism.

[Item 22] The method according to Item 21, wherein the pathogenic microorganism is a DNA virus or an RNA virus.

[Item 23] The method according to Item 22, wherein the RNA virus is an RNA virus having an envelope.

[Item 24] A group in which the RNA virus having an envelope consists of a flavivirus family virus; a togavirus family virus; a coronavirus family virus; an orthomixovirus family virus; a bunyavirus family virus; a paramyxovirus family virus; and a phyllovirus family virus. Item 23. The method according to Item 23, which is at least one selected from the above.

Item 25. The method according to Item 23 or 24, wherein the RNA virus having an envelope is a coronaviridae virus.

[Item 26] The coronavirus family virus is at least one selected from the group consisting of SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, and SARS-nCOV-2 coronavirus. Item 24 or 25.

[Item 27] The method according to Item 22, wherein the RNA virus is an RNA virus having no envelope.

[Item 28] The RNA virus having no envelope is at least one selected from the group consisting of astroviridae virus; caliciviridae virus; picornaviridae virus; hepeviridae virus; and leoviridae virus. 27. The method of item 27.

29. The method of item 27 or 28, wherein the non-enveloped RNA virus is norovirus or rotavirus.

[Item 30] The method according to any one of Items 27 to 29, wherein the RNA virus having no envelope is a norovirus, and it is possible to determine whether the norovirus is a GI type or a GII type.

31. The method of any of Items 1-30, wherein the PCR reaction solution further comprises at least one selected from the group consisting of bovine serum albumin and gelatin.

Item 32. The method according to any one of Items 1 to 31, wherein the PCR reaction solution further contains 1 mM or more of divalent cations.

[Item 33] A sample not subjected to nucleic acid isolation treatment, which comprises a visible dye used for preparing a sample mixture and a PCR reaction solution containing a DNA polymerase having contamination resistance. A test kit or composition used to test for the presence or absence of two or more target nucleic acids in two or more fluorescent compounds.

34. The kit or composition according to Item 33, wherein the DNA polymerase having contamination resistance is a DNA polymerase having reverse transcription activity.

Item 35. The kit or composition according to Item 33, wherein the PCR reaction solution further contains reverse transcriptase.

Item 36. The kit or composition according to any one of Items 33 to 35, wherein the DNA polymerase having contamination resistance is a DNA polymerase belonging to Family A.

Item 37: The DNA polymerase having contamination resistance is a DNA polymerase having at least one contaminant resistance selected from the group consisting of Tth, Howk Z05 and variants thereof. The kit or composition according to any of 36.

[Item 38] A DNA in which the mutant comprises an amino acid sequence exhibiting 90% or more identity with the amino acid sequence of Tth polymerase (SEQ ID NO: 25) or Hawk Z05 polymerase (SEQ ID NO: 26) and having contamination resistance. Item 6. The kit or composition according to Item 37, which exhibits polymerase activity.

Item 39 The variant comprises an amino acid sequence having one or several amino acid deletions, substitutions and / or additions in the amino acid sequence of Tth polymerase (SEQ ID NO: 25) or Hawk Z05 polymerase (SEQ ID NO: 26). The kit or composition according to Item 37 or 38, which exhibits DNA polymerase activity having contamination resistance.

[Item 40] The reverse transcriptase is a reverse transcriptase derived from at least one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and variants thereof. The kit or composition according to any one of 35 to 39.

[Item 41] The visible dye is purple (400-420 nm), indigo color (420-440 nm), blue (440-490 nm), green (490-570 nm), yellow (570-585 nm), orange (585-620 nm). 33-40. The kit or composition according to any one of Items 33 to 40, which is a dye exhibiting a color tone (maximum absorption wavelength in parentheses) selected from the group consisting of red (620-780 nm), white, black, and gray. thing.

[Item 42] Visible pigments are amaranth, erythrosin, alla red, ponceau, phenol red, orange G, ponso S, cresol red, rose bengal, tartrazine, sunset yellow, fast green, bromocresol green, brilliant blue, indigo. Item 6. The kit or composition according to any one of Items 33 to 41, which is at least one selected from the group consisting of carmine, patent blue, bromophenol blue, and bromocresol purp.

[Item 43] As the visible dye, a visible dye-containing solution in which the visible dye is suspended in at least one solvent selected from the group consisting of water, physiological saline, a buffer solution, an acidic solution, an alkaline solution, and an organic solvent. The kit or composition according to any one of Items 33 to 42, which comprises.

[Item 44] The kit or composition according to any one of Items 33 to 43, wherein the concentration of the visible dye in the visible dye-containing solution is 0.00001% or more.

Item 45. The kit or composition according to any one of Items 33 to 44, wherein the final concentration of the visible dye in the PCR reaction solution at the time of the PCR reaction is adjusted to 0.000001% or more.

[Item 46] The kit or composition according to any one of Items 33 to 45, wherein at least one of the fluorescent compounds is a double-stranded DNA-bound fluorescent compound.

[Item 47] Double-stranded DNA-bound fluorescent compounds are SYBR® Green I, SYBR® Gold, SYTO-9, SYTP-13, SYTO-82, EvaGreen®, LCGreen, Item 4. The kit or composition according to Item 46, which is one or more selected from the group consisting of Light Cycler (registered trademark) 480 ResoLight.

Item 48. The kit or composition according to any one of Items 33 to 47, wherein at least one of the fluorescent compounds is a hybridization probe.

Item 49. The kit or composition according to Item 48, wherein the hybridization probe is a TaqMan probe.

[Item 50] The kit or composition according to any one of Items 33 to 49, wherein the two or more kinds of fluorescent compounds include a combination of a hybridization probe and a double-stranded DNA-bound fluorescent compound.

According to the present invention, a sample that has not been isolated from microbial nucleic acid and may contain contaminants and insoluble substances is mixed with a visible dye to prepare a sample mixture, which is then added to the reaction solution for PCR reaction or RT-. By performing a PCR reaction, even when detecting two or more target nucleic acids, it is possible to inspect with sufficient sensitivity. By mixing the sample and the visible dye to obtain a pre-colored sample mixture, it is possible to suppress a mistake in adding the sample, which further improves the efficiency of genetic testing and pathogenic microorganism testing. Improving the efficiency of pathogenic microorganism testing work can increase the amount of testing for subjects who do not develop symptoms even if they are infected, and can greatly contribute to the prevention of infectious diseases. In addition, by pre-coloring a sample containing a sample that may be infectious with a visible dye, it is easy to visually check even in situations where there is a concern that attention may be reduced, for example, when handling a large number of samples. It may also be possible to call attention to handling that can be discriminated.

It is a figure which shows the example of the multiplex detection of norovirus RNA by the two enzyme system 1 step RT-PCR in the presence of various visible dyes. It is a figure which shows the example of multiplex detection (two enzyme system 1 step RT-PCR) of SARS-CoV-2 coronavirus sample in the presence of saliva sample and various visible dyes. It is a figure which shows the example of the multiplex detection (two enzyme system 1 step RT-PCR) of a saliva sample using various DNA polymerases and a SARS-CoV-2 coronavirus sample in the presence of a visible dye.

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention, but the present invention is not limited thereto. It should be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted.

In addition, all of the non-patent documents and patent documents described in the present specification are incorporated herein by reference. "-" In the present specification means "greater than or equal to, less than or equal to", and for example, if "X to Y" is described in the present specification, it means "more than or equal to X, less than or equal to Y". In addition, "and / or" in the present specification means either one or both. It should also be understood herein that the singular representation also includes the concept of its plural, unless otherwise noted.

One aspect of the present invention is, for example, an examination of pathogenic microorganisms (including viruses, bacteria, fungi, etc.) in a sample, in which nucleic acids (DNA, RNA) of pathogenic microorganisms are isolated and purified from the sample. After preparing a colored sample mixture by mixing a sample containing a contaminating substance with a visible dye, a PCR reaction solution (including an RT-PCR reaction solution) containing a DNA polymerase having contamination resistance is added to cause pathogenicity. It is a method for examining the presence of a pathogenic microorganism, which comprises detecting two or more different target nucleic acids derived from a sex microorganism with two or more kinds of fluorescent compounds.

The method for inspecting the presence or absence of two or more target nucleic acids in the sample of the present invention with one PCR reaction solution is characterized by including at least the following steps.

(1) A step of preparing a sample mixture containing a sample that has not been isolated from nucleic acid and a visible dye.

(2) A step of mixing the sample mixture prepared in the above step (1) with a PCR reaction solution containing a DNA polymerase having contamination resistance;

(3) A step of carrying out a PCR reaction after sealing the reaction vessel; and

(4) A step of detecting two or more target nucleic acids with two or more types of fluorescent compounds.

Here, in the step (1), after preparing the sample mixture containing the visible dye, a solution for pretreatment may be added separately, or the visible dye is suspended in the solution for pretreatment as the visible dye. A visible dye-containing solution may be used. Further, a pretreatment step such as heating may be included. When the pretreatment is performed in the step (1), it may be allowed to stand at room temperature for 1 second or longer, or may be heated at 70 ° C. or higher for 1 second or longer. In the step (2), the PCR reaction solution may be an RT-PCR reaction solution in which a reverse transcription reaction is also performed before the PCR reaction. Further, in the step (2), an RT-PCR reaction accompanied by a reverse transcription reaction may be performed before the PCR reaction. When the RT-PCR reaction is carried out in the step (2), even if it is a two-enzyme reaction system containing both reverse transcriptase and DNA polymerase, it is a one-enzyme reaction system containing a heat-resistant DNA polymerase having reverse transcription activity. You may. It is preferable that the steps (2) to (4) are performed in the same container. That is, it is preferable not to transfer all or a part of the mixed solution to another container during any of the steps (2) to (4). Further, in the step (3), preferably in both the steps (3) and (4), it is preferable not to open or close the lid of the reaction vessel after sealing the reaction vessel. Further, the sample that can contain the contaminating substance and the insoluble substance used in the step (1) may be a sample as it is collected from the subject, or a suspension suspended in water or a buffer solution in advance. It may be a liquid, and a solid sample such as a stool sample may be added directly to the solution containing the visible dye.

The test target in the present invention may be, for example, a nucleic acid derived from a pathogenic microorganism, but is not particularly limited. Here, the pathogenic microorganism refers to any microorganism that can infect living organisms such as humans and other mammals and cause infectious diseases, and is not limited to prokaryotes such as bacteria or fungi and eukaryotes. It is a concept that includes viruses and the like.

Examples of pathogenic microorganisms include Escherichia coli (eg, enterohemorrhagic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EIEC), toxinogenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EAEC), enterohemorrhagic Escherichia coli (EHEC)). ; Campylobacter, Welsh, Salmonella, Listeria, Botulinum, Seleus, Pseudomonas (multi-drug resistant Escherichia coli), Clostridium, Rezionera, Streptococcus, yellow Escherichia coli (eg, methicillin-resistant Staphylococcus aureus), Acinetobacta spp. Examples thereof include Staphylococcus aureus, Chlamydia, Escherichia coli, Toxoplasma, etc., but are not particularly limited. In particular, the detection of pathogens that cause food poisoning such as Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella may be performed as an intestinal bacterial test that handles a large number of samples, and is excellent in workability. A simple inspection method of the present invention is useful.

In certain embodiments, the pathogenic microorganism may be a virus. The virus to be the subject of the present invention may be a DNA virus or an RNA virus. DNA viruses include herpesviridae virus (eg, simple herpesvirus, cytomegalovirus); adenoviridae virus (eg, human adenovirus); hepadonaviridae virus (eg, hepatitis B virus); papovaviridae virus. (Eg, papillary virus, polyomavirus); parvoviridae virus (eg, adeno-associated virus, human parvovirus); asfaviridae virus (eg, African pig fever virus), etc., but are particularly limited. It's not a thing.

The RNA virus may be an RNA virus having no envelope derived from the lipid double membrane or an RNA virus having an envelope. Such non-enveloped RNA viruses include astroviridae virus (eg, astrovirus); caliciviridae virus (eg, sapovirus, norovirus); picornaviridae virus (eg, hepatitis A virus, echovirus, etc.). Enterovirus, coxsackie virus, poliovirus, rhinovirus); hepevirus family virus (eg, hepatitis E virus); leovirus family virus (eg, rotavirus), etc., but not limited to, preferably. It is useful for the detection of Calisivirus and Leovirus viruses, more preferably for the detection of norovirus, sapovirus, and rotavirus, and particularly useful for the detection of norovirus. Most non-enveloped viruses can infect the gastrointestinal tract due to fecal-oral infection, etc., and RNA is retained in a rigid capsid structure that is resistant to inactivation by gastric acid and the surface-active action of bile acids.

Norovirus has a capsid structure in which the viral RNA genome is encapsulated inside an icosahedron consisting of a capsid protein of about 30 nm. The capsid structure is resistant to inactivation by gastric acid and the surface-active action of bile acids so that the virus can survive in harsh environments such as the digestive tract. A virus inactivating agent such as a normal surfactant or 70% ethanol cannot destroy this capsid structure, and the infectivity of the virus is maintained. In order to destroy the capsid structure, heat treatment under harsh conditions of at least 85 ° C. or higher for 1 minute or longer is required (Non-Patent Document 1), but RNA is extracted using a commercially available viral RNA extraction kit. It takes a lot of labor to purify.

The main cause of norovirus infection is eating and drinking foods contaminated with norovirus, but since infection is often caused by human hands, regular stool inspections are conducted at cooking facilities, medical sites, elderly care facilities, nurseries, etc. Is required. The mass cooking facility hygiene management manual has added that the stool test for cooks, etc. should include a norovirus test at least once a month or, if necessary, from October to March, which is the epidemic period of norovirus. .. This is because there are quite a few people (health carriers) who are infected with the virus but have no symptoms, and these people may unknowingly spread the infection. In addition, cooks with symptoms such as diarrhea and vomiting should consult a medical institution, and if it is found that they are infected with norovirus, perform high-sensitivity tests such as real-time PCR and do not have norovirus. It is desirable to take appropriate measures such as refraining from cooking work that comes into direct contact with food until it is confirmed. Therefore, it is of great significance to carry out regular tests including healthy people, and it is required to carry out tests for multiple samples. It is preferable that the present invention is used for norovirus testing from the viewpoint that even when a large number of samples are handled, the test can be performed easily and with good workability, and the burden on the person in charge of testing can be reduced.

Envelope RNA viruses include flaviviridae viruses (eg, hepatitis C virus, Japanese encephalitis virus, decavirus, pig fever virus); Togaviridae virus (eg, ruin virus, chikungnia virus); coronaviridae virus (eg, coronavirus family virus). , SARS Coronavirus, MERS Coronavirus, SARS-CoV-2 Coronavirus); Orthomixoviridae virus (eg, influenza virus); Rabdoviridae virus (eg, mad dog disease virus); Bunyaviridae virus (eg, Crimea congo) Fever virus, Hunter virus); Paramyxovirus family virus (eg, measles virus, human RS virus); Phyllovirus family virus (eg, Ebola virus), etc., but are not particularly limited, and particularly corona. Useful for virus detection.

Coronavirus is a causative virus that causes respiratory infections including colds, and it is said that about 10 to 35% of coronaviruses are caused by the coronavirus during the cold season. It is also known that mutant viruses occur, and rarely SARS (severe acute respiratory syndrome) coronavirus, MERS (Middle East respiratory syndrome) coronavirus, and new coronavirus infection (COVID-19) coronavirus (SARS). -It is known that those that cause fatal and serious respiratory diseases such as CoV-2) occur. Therefore, it goes without saying that simple, rapid, and highly sensitive detection of coronavirus is important in clinical diagnosis, food hygiene inspection, environmental inspection, and the like.

For pathogen testing of coronavirus, methods for detecting viral genes using electron microscopy, ELISA-based immunological antigen detection, or nucleic acid amplification techniques have been developed. Among these test methods, nucleic acid amplification technology capable of detecting coronavirus with high sensitivity is widely used. Several techniques have been developed for detecting coronavirus by nucleic acid amplification method (for example, Non-Patent Document 2, Non-Patent Document 3, Patent Document 2).

For the mutant coronavirus SARS-CoV-2, which was confirmed to occur in Wuhan City, Hubei Province, China in 2019, a test method using nucleic acid amplification technology was established as soon as the analysis of viral genomic RNA was completed (for example). , Non-Patent Document 3, Non-Patent Document 4). Also in Japan, a method for detecting SARS-CoV-2 is described in the "Pathogen Detection Manual 2019-nCoV" of the National Institute of Infectious Diseases (Non-Patent Document 5). In these techniques, detection of coronavirus contained in a sample involves extraction and purification of viral RNA from the sample. The process of extracting and purifying viral RNA was complicated and required a lot of working time. Currently, rapid 1-step RT-PCR is used without RNA extraction and purification steps from biological samples such as pharyngeal / nasal swabs containing coronavirus, especially SARS-CoV-2, saliva, sputum, and fecal samples, and wiping environment samples. It is desired to develop a method that can detect the virus. In addition, from the viewpoint of infection prevention and infection spread prevention, it is strongly desired to expand the test including asymptomatic persons, but in the multi-sample test, it is inevitable to add and dispense the sample including the sample. As the number increases, there is concern that the burden on the inspector will increase. Dispensing work in the inspection of such a large number of samples may cause mistakes such as dispensing omission and multiple dispensing. Since these mistakes cause re-inspection and the like, it is strongly desired to develop an inspection method with improved workability. In addition, keeping samples containing infectious samples that require careful handling in a state that can be easily visually identified from the early stages of inspection work also reduces the burden on the person in charge of inspection. Can be connected. Since the present invention can detect two or more target nucleic acids easily and with high sensitivity while being a method having excellent workability and operability, it is particularly effective in testing such coronaviruses, especially SARS-CoV-2 virus. It is beneficial.

Examples of the sample used in the present invention include, but are particularly limited, pharyngeal swab, nasal swab, sputum, feces (excretion, rectal stool), vomitus, saliva, mouthwash, tears, and the like. It can be used for all things derived from living organisms. In particular, it is useful for detection from feces, pharyngeal swabs, nasal swabs, sputum, lung aspirates, cerebrospinal fluid, mouthwash, saliva, tears, cultured cells, and culture supernatants, especially saliva samples. It is useful for detecting samples such as mouthwash, tear fluid, pharyngeal swab sample, and nasal swab sample, which are usually translucent and have low turbidity and tend to be difficult to see. One of the features of the present invention is that it is not necessary to isolate RNA from these samples using a commercially available RNA purification kit or the like. The sample may be subjected to direct detection, or the sample may be suspended in water, saline or buffer in order to reduce the influence of impurities on the reaction and obtain more stable test results. There may be. The buffer solution is not particularly limited, and examples thereof include Hanks buffer solution, Tris buffer solution, phosphate buffer solution, glycine buffer solution, HEPES buffer solution, and tricine buffer solution. Further, in the case of a highly viscous biological sample (for example, a sample containing highly viscous sputum), the sample may be a sample treated with a sputazyme enzyme solution, although not particularly limited.

Another example of the sample in the present invention is a wipe inspection sample. Wiping inspection is useful for clarifying the pollution route and grasping the pollution status such as the facility environment. In the present invention, the wiping test is not particularly limited, but is a sample obtained by wiping the relevant section or equipment with a cotton swab or the like, eluting into water or a buffer solution, and concentrating with polyethylene glycol (PEG) precipitate or the like. .. As a specific procedure for the wiping test, "improvement of the norovirus test method for the wiped sample" (http://idsc.nih.go.jp/iasr/32/382/dj3824.html) is exemplified. There is no particular limitation, and methods similar to this are widely included. Examples of wipes include cutting boards, kitchen knives, kitchen utensils, tableware and other cooking utensils, refrigerator handles and toilets, bathroom door knobs, washrooms, kitchens, toilets, bathroom faucets, cookers' hands and fingers, and bathrooms. , Toilet, washbasin, handrail, living room and other facilities. Although it is not a wiping test, it can also be applied to a concentrated sample of a sewage sample as an environmental test. Since these test samples contain a large amount of dirt and dust at the test site, this method with enhanced contamination resistance in samples that may contain contaminants and insoluble substances is useful for these tests.

In certain embodiments, the method of the invention is characterized by the use of a sample that has not been treated with nucleic acid isolation. For example, the present invention may isolate nucleic acids from various samples with a commercially available nucleic acid purification kit, or may undergo prior heat treatment to expose genomic nucleic acids from the structure of pathogenic microorganisms (eg, cell membranes, capsid structures). Untreated samples can be used. It is preferable to use these untreated samples from the viewpoint of simplicity because it eliminates the need for time-consuming pretreatment. Further, the sample may be a sample obtained by nucleic acid extraction without separation and purification to remove contaminants. Here, the sample from which nucleic acid extraction is performed without separation and purification means that the nucleic acid is exposed in the sample, for example, the cell membrane, capsid, envelope, etc. are destroyed in the sample, and these are used. Extracting and exposing the encapsulated nucleic acid (however, do not remove the cell membrane, capsid, envelope fragment, etc. remaining after destruction). In the present invention, nucleic acid can be satisfactorily amplified even in a sample prepared without the trouble of isolation and purification, and a stable test result can be obtained. The nucleic acid extraction process that does not involve such separation and purification can be performed prior to the step (1).

Contaminants and insoluble substances that can be contained in the sample to be subjected to PCR or RT-PCR in the step (1) include feces (excretion stool, rectal stool), vomitus, saliva, sputum, mouthwash, nasal swab, and pharynx. Examples include those derived from wipes, tears, blood, and wiping test samples, but are not limited to those derived from living organisms and can be used for all environmental test samples, especially feces (excretion). It is useful for detection from feces, rectal feces), saliva, sputum, mouthwash, tears, pharyngeal swab, and nasal swab. The concentration of insoluble substances that can be contained varies depending on the test sample, but if the turbidity OD660 is contained in the PCR reaction solution or the RT-PCR reaction solution, for example, 0.01 Abs / μL or more, the test sensitivity may be affected. However, it is not limited to this. For example, the turbidity OD660 may be 0.05 Abs / μL or more, 0.1 Abs / μL or more, 0.5 Abs / μL or more, and 1 Abs / μL or more, but is not particularly limited. Further, as long as the effect of the present invention is exhibited, the upper limit of the concentration of the insoluble substance that can be contained is not particularly limited, but may be, for example, 5 Abs / μL or less and 3Abs / μL or less, but is not limited. According to the present invention, even if a sample mixture having a changed color tone is prepared by mixing a high turbidity test sample with a visible dye and then mixed with a PCR reaction solution and reacted, two or more kinds are prepared. It may be possible to detect more than one target nucleic acid with the fluorescent compound of.

The work of purifying nucleic acid derived from a microorganism from a sample causes the work to be complicated and the work time to be extended. In addition to this, the work of adding a sample into a reaction vessel such as a PCR tube or a PCR plate can be hundreds or thousands of times depending on the number of samples. The continuous addition to the reaction vessel and the execution of the dispensing operation may cause mistakes such as omission of addition to the reaction vessel and multiple additions. These mistakes make it impossible to carry out the inspection correctly, and further work is required due to re-inspection, etc., resulting in time and financial loss. In the present invention, in the inspection of such pathogenic microorganisms, by simplifying the work at the work site and enabling the inspection to be carried out promptly, it is possible to prevent further spread of infection. Therefore, improving the efficiency of the dispensing work by mixing the visible dye and the sample to obtain a colored sample mixture and adding this sample mixture to the PCR reaction solution has more significance than improving the work efficiency. There is.

The visible dye is a dye having a spectral sensitivity that can be detected under white light in the visible region. By mixing or dissolving the visible dye in the solution, the solution can be given a visually perceptible color. Visible dyes are preferably visually identifiable with the naked eye. When the solution is colored with a visible dye, it is not particularly limited as long as it can be visually identified, but it is desirable that the effect on the detection system using the fluorescent dye is not too large. In a preferred embodiment, it is preferred to use an aqueous visible dye to color the solution.

The colored solution has an absorption spectrum in at least one wavelength range. The color tone of visible dyes is classified by the maximum absorption wavelength. The color tones of the visible dye used in the present invention (maximum absorption wavelength in parentheses) are purple (400-420 nm), indigo (420-440 nm), blue (440-490 nm), green (490-570 nm), and yellow. (570-585 nm), orange (585-620 nm), red (620-780 nm), and shades of white to black (eg, gray) are also included, but are not particularly limited as long as they are visually perceptible. .. Preferably, purple (400-420 nm), indigo (420-440 nm), blue (440-490 nm), green (490-570 nm), yellow (570-585 nm), orange (585-620 nm), red (620-780 nm). ) Is preferred, purple (400-420 nm), indigo (420-440 nm), green (490-570 nm), yellow (570-585 nm), orange (585-620 nm), red (85-620 nm). It is more preferable to use a visible dye having a color tone of 620-780 nm), and it is further preferable to use a visible dye having a color tone of orange (585-620 nm) and red (620-780 nm). As shown in the results of the test examples described later, by using a visible dye exhibiting such a color tone, two or more kinds of fluorescent compounds (for example, known fluorescent compounds such as FAM, Cy5, ROX, and HEX) can be used. Even when the above target nucleic acids are detected, highly sensitive detection may be possible.

Visible dyes used in the present invention include, but are not limited to, for example, amaranth, erythrosine, alla red, ponceau (Acid red18), phenol red, orange G, ponso S, cresol red, rose bengal, tartrazine. , Sunset Yellow, Fast Green, Bromocresol Green, Brilliant Blue, Indigo Carmine, Patent Blue, Bromophenol Blue (BPB), Bromocresol Purp and the like. It may contain a single visible dye, or by mixing a plurality of visible dyes, it is possible to use it as a color tone different from that when the single visible dye is used. In addition, some visible dyes have the property that the maximum absorption wavelength and color tone change depending on the hydrogen ion concentration (pH) in the solution. It is preferable to select the visible dye to be used according to the pH of the reaction solution.

The absorption spectrum of the solution depends on the concentration of the visible dye as well as the type of visible dye that is dissolved. The dye concentration is represented by the abundance ratio (% w / w) of the dye in the solution, the absorbance at the maximum absorption wavelength (optical path length 1 mm), and the like. The concentration of the visible dye used in the present invention is not particularly limited, but is, for example, 1% w / w or less, preferably 0.1%, based on the whole sample mixture after mixing with the sample not subjected to the isolation treatment. It is w / w or less, more preferably 0.01% w / w or less, still more preferably 0.001% w / w or less, still more preferably 0.0001% w / w or less. The lower limit is not particularly limited, but may be, for example, 0.000000001% w / w or more with respect to the entire sample mixture. The maximum absorption wavelength is not particularly limited, but the absorbance is 0.5 or less, more preferably 0.1 or less, and even more preferably 0.01 or less. The lower limit of the maximum absorption wavelength is also not particularly limited, but may be, for example, 0.0001 or more. The concentration of the visible dye in the entire PCR reaction solution after mixing with the PCR reaction solution is also not particularly limited, and is, for example, 0.1% w / w or less, more preferably 0.01% w / w or less, still more preferably 0. It can be 001% w / w or less, even more preferably 0.0001% w / w or less, even more preferably 0.00001% w / w or less. The lower limit is not particularly limited, but may be, for example, 0.00000001% w / w or more with respect to the entire PCR reaction solution.

In the step (1), the visible dye may be solid, for example, powder or fine powder, or may be arbitrary such as water, physiological saline, buffer solution, acidic solution, alkaline solution, and organic solvent. It may be a visible dye-containing solution in which the visible dye is suspended in the solvent of. It is preferable to use a visible dye-containing solution in which the visible dye is suspended in a solvent such as a buffer solution, an acid or alkaline solution, or an organic solvent. Nucleic acid-unisolated samples are premixed with buffers, acid or alkaline solutions, organic solvents and other solutions to facilitate the extraction of RNA or DNA from robust structures such as capsids and cell walls. After mixing the visible dye with the sample, an additional solution having these properties may be added. The acidic solution is not particularly limited as long as it is an acidic solution. Examples of the acidic solution include formic acid aqueous solution, acetic acid aqueous solution, butyric acid aqueous solution, hydrochloric acid aqueous solution, nitrate aqueous solution, sulfuric acid aqueous solution, citric acid aqueous solution, lactic acid aqueous solution, phosphoric acid aqueous solution, benzoic acid aqueous solution, oxalic acid aqueous solution, tartaric acid aqueous solution, and ascorbic acid aqueous solution. , Sulphonic acid aqueous solution and the like, and can be used alone or in combination of two or more. The alkaline solution is not particularly limited as long as it is an alkaline solution. Examples of the alkaline solution include potassium hydroxide aqueous solution, sodium hydroxide aqueous solution, lithium hydroxide aqueous solution, magnesium hydroxide aqueous solution, calcium hydroxide aqueous solution, barium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, magnesium carbonate aqueous solution, and calcium carbonate. Aqueous solution, Tris buffer, glycine buffer, phosphate buffer, borate buffer, Good buffer (TAPSO, POPSO, HEPSO, EPPS, Tricine, Bicine, TAPS, CHES, CAPS), etc. Or it can be used in combination of two or more. Specific examples of the organic solvent include ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, triethylamine, dimethylformamide, hexamethylphosphoric triamide, dimethylsulfoxide, acetone, acetonitrile, ethanol and methanol. , 1-propanol, 2-propanol, 1-butanol, pyridine and the like, but are not limited thereto. Further, the sample added in the step (1) may be one that has been subjected to heat treatment after being mixed with the solution. The conditions of the heat treatment are not particularly limited, but may be those treated at 60 ° C. or higher, preferably 70 ° C. or higher, more preferably 80 ° C. or higher, still more preferably 90 ° C. or higher for 1 second or longer.

When a visible dye-containing solution in which the visible dye is suspended in an arbitrary solvent such as water, physiological saline, a buffer solution, an acidic solution, an alkaline solution, or an organic solvent is used as the visible dye, the concentration thereof has the effect of the present invention. It is not particularly limited as long as it is played. As an example, the concentration of the visible dye in the visible dye-containing solution is 3% w / w or less, preferably 1% w / w or less, more preferably 0.5% w / w or less, still more preferably 0.1%. It can be w / w or less, even more preferably 0.05% w / w or less, and may be, for example, 0.01% w / w or less. The lower limit is not particularly limited, but for example, 0.00001% w / w or more, preferably 0.0001% w / w or more, and more preferably 0.001% w / w or more with respect to the entire visible dye-containing solution. Can be.

The PCR cycle in the step (3) is 1. Heat treatment and 2. It may include a step of reverse transcription reaction. In addition, a heat treatment step for activating the hot start enzyme may be included before and after each step. The heat treatment step 1 may include disrupting the virus to expose the nucleic acid in the virus and / or activating the hot start enzyme in the nucleic acid amplification reaction. The temperature and time of the heat treatment step may be 60 ° C. or higher and 1 second or longer, preferably 70 ° C. and 30 seconds or longer, more preferably 80 ° C. and 30 seconds or longer, and particularly preferably 85 ° C. and 30 seconds. More than a second. The temperature of the reverse transcription reaction of 2 is determined by the reverse transcriptase activity of the reverse transcriptase used and the Tm value of the primer and the probe, and may be at least 25 ° C. or higher. More preferably, it is 37 ° C. or higher. In PCR of 3, [1] DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), [2] annealing of primers to template single-stranded DNA, and [3] the above-mentioned using DNA polymerase. It suffices to include three steps of primer extension, and [2] and [3] may be carried out at the same temperature to form two steps. In order to carry out rapid RT-PCR, the thermal cycler used for the RT-PCR reaction has a total extension time of the steps [2] and [3] of 15 seconds or less, more preferably 10 seconds or less. It is desirable to set the measurement program of. In the present specification, the "PCR extension time" refers to the set temperature in the thermal cycler.

In the first step step (2), the sample mixture is mixed with a PCR reaction solution containing a DNA polymerase having contamination resistance. As the DNA polymerase contained in the PCR reaction solution, any DNA polymerase known in the art can be used as long as it has resistance to contaminants. Contaminant resistance refers to the property of having high enzymatic activity of DNA polymerase sufficient for nucleic acid amplification reaction even in the presence of PCR inhibitor. DNA polymerases having resistance to contaminants include, but are not limited to, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, HowkZ05 and variants thereof. However, it is not particularly limited. Preferably, Tth (SEQ ID NO: 25) and HawkZ05 (SEQ ID NO: 26) or variants thereof are used. Particularly preferred is the use of Tth or a variant thereof. Even a DNA polymerase such as Taq, which normally does not have contamination resistance, can be used if it is a mutant having contamination resistance due to an amino acid mutation. As an example, the total amount of the DNA polymerase having resistance to contaminants contained in the PCR reaction solution may be at least 4.2 ng / μL or more, preferably 5.0 ng / μL or more, and 5.8 ng / μL. The above is more preferable. Above all, it is preferably 8.3 ng / μL or more. The upper limit of the total amount of the DNA polymerase having contamination resistance is not particularly limited, but as an example, it can be 20 ng / μL or less, and even if it is 16.7 ng / μL or less, the effect of the present invention can be sufficiently obtained. Can be done. The amount of polymerase is a value quantified by the Bradford method or Nanodrop (Thermo Fisher), and may be estimated from the Safety Data Sheet (SDS). When a protein such as BSA is contained, it is desirable to calculate by the latter method. In order to enhance the effect of suppressing non-specific reaction, the enzymatic activity of DNA polymerase until the start of PCR reaction is suppressed by introducing it into DNA polymerase of heat unstable block group in combination with anti-DNA polymerase antibody or by chemical modification. , It is preferable that it can be applied to hot start PCR.

When the step of the reverse transcription reaction is included in the step (3), the PCR solution added to the mixed solution contains DNA polymerase and, if necessary, reverse transcriptase. The origin of the reverse transcriptase contained in the PCR reaction solution is not particularly limited as long as RNA can be converted into DNA, but is MMLV (Moloney Murine Leukemia Virus) -RT, AMV-RT (Avian Myeloblastosis Virus), HIV-RT, RAV2. -RT, EIAV-RT, Carboxydothermus hydrogenoformam DNA polymerase) and variants thereof are exemplified. Particularly preferred examples include MMLV-RT, AMV-RT, or variants thereof.

The reverse transcriptase may be a DNA polymerase having both reverse transcriptase activity. The DNA polymerase having reverse transcription activity is a DNA polymerase having both the ability to convert RNA into cDNA and the ability to amplify DNA. A DNA polymerase having reverse transcription activity is preferably thermostable in addition to contamination resistance. The heat resistance means that the enzyme activity does not decrease by more than half even if the heat treatment is carried out at 70 ° C. for 1 minute or more. The origin is not particularly limited, and examples thereof include Taq, Tth, Bst, Bca, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and variants thereof. As DNA polymerases having reverse transcription activity so far, DNA polymerase derived from Thermus aquaticus (Taq), DNA polymerase derived from Thermus thermophilus HB8 (Tth), DNA polymerase derived from Thermus sp Z05 (Z05), and DNA polymerase derived from Thermotoga maritima. (Tma), DNA polymerase (Bca) derived from Bacillus caldotenax, DNA polymerase (Bst) derived from Bacillus theatermophilus, etc., even if these variants do not lose their reverse transcription activity and heat resistant DNA polymerase activity. good. Further, a variant of DNA polymerase (KOD) derived from Thermococcus kodakaraensis is known to have reverse transcriptase activity (for example, RTX: reverse transcription xenopolymerase), and such reverse transcriptase activity is described in the present invention. A heat-resistant DNA polymerase having both can also be used. Particularly preferred are DNA polymerases having reverse transcription activity selected from the group consisting of Tth, Z05 and variants thereof.

In the present specification, the variant of DNA polymerase having contamination resistance is, for example, 85% or more, preferably 90% or more, more preferably 95% or more with respect to the amino acid sequence of the wild-type DNA polymerase from which it is derived. Further preferably, it has 98% or more, particularly preferably 99% or more of sequence identity, and has high DNA polymerase activity even in the presence of contaminants. When it is a DNA polymerase that also has reverse transcription activity, it means an activity that has an activity of converting RNA into cDNA and an activity of amplifying DNA even in the presence of a contaminating substance. Here, as a method for calculating the identity of the amino acid sequence, any means known in the art can be used. For example, it can be calculated using analysis tools available on the market or through telecommunications lines (Internet), for example, the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http. : // www. ncbi. nlm. nih. By using the default (initial setting) parameters in gov / BLAST /, it is possible to calculate the identity of the amino acid sequence. Further, in the mutant that can be used in the present invention, one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of the wild-type DNA polymerase from which the mutant is derived (hereinafter, these are collectively referred to as “). It may be a polypeptide consisting of an amino acid sequence (also referred to as "mutation"), and may have an activity of converting RNA into DNA and an activity of amplifying DNA as in the case of wild-type DNA polymerase. Here, 1 or several may be, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, and even more preferably 1 to 5, but the number is not particularly limited.

In addition to DNA polymerase, the PCR reaction solution used in the present invention includes a buffer, a suitable salt, a magnesium salt or a manganese salt, a deoxynucleotide triphosphate, a detection target region of a nucleic acid derived from a virus to be detected or a pathogenic microorganism. The corresponding primer pair and, if necessary, an additive may be contained.

The buffer used in the present invention is not particularly limited, and examples thereof include Tris, Tricine, Bis-Tricine, and Bicine. The pH was adjusted to 6 to 9, more preferably pH 7 to 9 with sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid or the like. The concentration of the buffer to be added is 10 to 200 mM, more preferably 20 to 150 mM. At this time, a salt solution is added in order to obtain ionic conditions suitable for the reaction. Examples of the salt solution include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate and the like.

As the dNTP used in the present invention, dATP, dCTP, dGTP, and dTTP are added at 0.1 to 0.5 mM, respectively, and most commonly, about 0.2 mM is added. Prophylactic measures against cross-contamination may be taken by using dUTP as an alternative and / or as part of dTTP. When taking preventive measures against cross-contamination, it is preferable to include Uracil-N-gycosylase (UNG).

Further, in the present invention, it is preferable that the PCR reaction solution contains divalent cations. By including divalent cations in this way, more stable and high contamination resistance can be obtained, and highly sensitive detection becomes possible. The divalent cation is not particularly limited, and examples thereof include magnesium ion, manganese ion, calcium ion, copper ion, iron ion, nickel ion, and zinc ion. It is preferable to include magnesium ion and manganese ion as the divalent cation. In the present invention, when magnesium ions, manganese ions, or the like are added to the PCR reaction solution, magnesium, manganese, or the like may be added, or salts thereof may be added. Examples of magnesium or a salt thereof include magnesium, magnesium chloride, magnesium sulfate, magnesium acetate and the like, and examples of manganese or a salt thereof include manganese, manganese chloride, manganese sulfate, manganese acetate and the like. It is preferable that such magnesium, manganese, or a salt thereof is added to the PCR reaction solution in an amount of about 1 to 10 mM. When performing monoenzyme-based RT-PCR in the test method of the present invention, it is preferable to contain manganese or a salt thereof from the viewpoint that stable and high sensitivity can be easily obtained. In certain embodiments, the RT-PCR reaction solution preferably contains 1 mM or more of manganese or a salt thereof, preferably 1.5 mM or more of manganese or a salt thereof, and 2.0 mM or more of manganese or a salt thereof. It is more preferable to include it. Further, when performing bienzyme-based RT-PCR or PCR without reverse transcription reaction, it is preferable to contain magnesium or a salt thereof from the viewpoint that stable and high sensitivity can be easily obtained. In certain embodiments, the PCR reaction solution preferably contains 1 mM or more of magnesium or a salt thereof, preferably 1.5 mM or more of magnesium or a salt thereof, and preferably contains 2.0 mM or more of magnesium or a salt thereof. Is more preferable.

Further, as an additive contained in the PCR reaction solution, a quaternary ammonium salt having a structure in which three methyl groups are added to an amino group in an amino acid (hereinafter referred to as "betaine-like quaternary ammonium"), and a polypeptide (bovine serum). It may contain at least one selected from the group consisting of albumin, sericin, Blocking peptide fragment (hereinafter also referred to as BPF), gelatin), glycerol, glycol and a surfactant.

The polypeptide used in the present invention is not particularly limited as long as it has a molecular weight of 5 to 500 kDa, but is preferably 6 to 400 kDa. In the present specification, when indicating a molecular weight, it means a value determined by using SDS-PAGE unless it is clear that it has another meaning. The measurement of the molecular weight by SDS-PAGE can be performed by using a method and an apparatus common in the art and using a commercially available molecular weight marker or the like. For example, "molecular weight 50 kDa" means that when the molecular weight is measured by SDS-PAGE, those skilled in the art usually determine that there is a band at the position of 50 kDa. Further, the polypeptide used in the present invention may be a mixture of polypeptides within the above molecular weight range.

The polypeptide used in the present invention is not particularly limited as long as it exerts the effect of the present invention, and refers to a protein formed by connecting a plurality of amino acids by peptide bonds. Further, the polypeptide used in the present invention is, for example, a heat-denatured polypeptide (for example, gelatin) whose three-dimensional structure is solved by heat denaturation or the like as long as it has a polypeptide structure in which amino acids are linked. You may. Specifically, the polypeptides that can be used in the present invention include, for example, albumin (eg, bovine serum albumin, lactoalbumin, human serum albumin, egg-derived albumin), gelatin (eg, fish gelatin, pig gelatin), sericin, and the like. Naturally-derived proteins such as casein and fibroin (naturally-derived polypeptides); artificially produced by synthesis / degradation of Blocking peptide fragment (hereinafter also referred to as BPF), collagen hydrolysate, polypeptone, yeast extract, beef extract, etc. Polypeptides and the like can be used. From the viewpoint that even more excellent effects of the present invention are exhibited, the polypeptides used in the present invention are preferably bovine serum albumin, gelatin, Blocking peptide fragment (hereinafter referred to as BPF), and / or sericin. From the viewpoint that a high effect can be exhibited even in a small amount, it is more preferable to use bovine serum albumin and gelatin (particularly fish gelatin). These polypeptides may be used alone or in combination of two or more. Further, these polypeptides may be prepared by means such as extraction from nature or synthesis, and commercially available products can also be preferably used.

In the present invention, the amount of the polypeptide used is not particularly limited as long as the effect of the present invention is exhibited, but for example, in a mixed solution of a sample containing the insoluble substance and a one-enzyme system 1-step RT-PCR reaction solution. In an amount such that the final concentration is 0.0001 to 200 mg / mL, preferably 0.01 to 150 mg / mL, more preferably 0.1 to 130 mg / mL, and further preferably 0.5 to 100 mg / mL. can do. The preferable amount for exerting a more excellent effect may vary depending on the type of polypeptide used, the degree of desired effect, and the like, and for example, the following amounts can be exemplified.

-When bovine serum albumin is used: The final concentration in the PCR reaction solution is, for example, 0.5 mg / mL or more, preferably 1 mg / mL or more, more preferably 2 mg / mL or more, still more preferably 3 mg / mL or more. The upper limit is not particularly limited, but can be, for example, 10 mg / mL or less.

-When gelatin is used: The final concentration in the PCR reaction solution is, for example, 0.1 mg / mL or more, preferably 1 mg / mL or more, more preferably 5 mg / mL or more, still more preferably 7.5 mg / mL or more, still more preferably. Is 15 mg / mL or more. The upper limit is not particularly limited, but can be, for example, 50 mg / mL or less.

When sericin is used: The final concentration in the PCR reaction solution is, for example, 1 mg / mL or more, preferably 5 mg / mL or more, more preferably 10 mg / mL or more, still more preferably 20 mg / mL or more, still more preferably 50 mg / mL. that's all. The upper limit is not particularly limited, but can be, for example, 100 mg / mL or less.

When BPF is used: The final concentration in the PCR reaction solution is, for example, 1 mg / mL or more, preferably 5 mg / mL or more, more preferably 10 mg / mL or more, still more preferably 20 mg / mL or more, still more preferably 30 mg / mL. that's all. The upper limit is not particularly limited, but can be, for example, 50 mg / mL or less.

Examples of the surfactant contained in the PCR reaction solution include Triton X-100 (Triton X-100), Triton X-114 (Triton X-114), Tween 20 (Tween 20), Nonidet P40, Briji35, Briji58, SDS, CHAPS, and the like. CHASPO, Emulgen 420 and the like can be mentioned, but the present invention is not particularly limited. The concentration of the surfactant in the PCR reaction solution is also not particularly limited, but is preferably 0.0001% or more, more preferably 0.002% or more, still more preferably 0.005% or more, and good detection is possible. Will be. The upper limit is not particularly limited, but as an example, it can be 0.1% or less.

Examples of the betaine-like quaternary ammonium contained in the PCR reaction solution include betaine (trimethylglycine) and carnitine, but are not particularly limited. The betaine structure is a compound having both positive and negative charges that is stable in the molecule, and exhibits properties like a surfactant, and is thought to cause destabilization of the virus structure. Furthermore, it is known to promote nucleic acid amplification of DNA polymerase. The preferred betaine-like quaternary ammonium concentration is 0.1 M to 2 M, more preferably 0.2 M to 1.2 M.

Furthermore, it can be used in combination with substances known in the art to promote PCR or RT-PCR. Accelerators useful in the present invention include, for example, glycerol, polyols, protease inhibitors, single strand binding proteins (SSBs), T4 gene 32 proteins, tRNA, sulfur or acetamide-containing compounds, dimethylsulfoxide (DMSO), glycerol, ethylene. Glycerol, Propylene Glycol, Trimethylene Glycol, Formamide, Acetamide, Ectoin, Trehalose, Dextran, Polyvinylpyrrolidone (PVP), Tetramethylammonium Chloride (TMC), Tetramethylammonium Hydroxide (TMAH), Tetramethylammonium Acetate (TMAA), Examples include, but are not limited to, polyethylene glycol. Ethyleneglycol-bis (2-aminoethyl ether) -N, N, N', N'-tetraacetic acid (EGTA), 1,2-bis (o-aminophenoxy) ethane-to further reduce reaction inhibition It may contain a chelating agent such as N, N, N', N'-tetraacetic acid (BAPTA).

In one embodiment, the method of the invention is characterized by a multiplex PCR that detects two or more target nucleic acids. Here, the "target nucleic acid" may be a nucleic acid region intended to be detected by nucleic acid amplification. For example, when examining the presence or absence of nucleic acid derived from a pathogenic microorganism, it may be a region intended for amplification in the genomic nucleic acid of the pathogenic microorganism. When the PCR reaction solution contains internal control and the like, the target nucleic acid may be a region intended for amplification in the internal control nucleic acid. The two or more target nucleic acids may be two or more nucleic acid regions in a region amplified by one primer pair, or may be a nucleic acid region in a region each amplified by two or more primer pairs. good. In the method of the present invention, the number of target targets is not particularly limited, but may be two or more, for example, three, four, five or more. The upper limit of the number of targets is not particularly limited, but may be, for example, 10 or less.

Primer pairs used in the present invention include two pairs of primers in which one primer is complementary to the DNA extension product of the other primer. Although not particularly limited, in the present invention, it is preferable that two or more pairs of the above primers are contained. In addition, if the target nucleic acid consists of subtypes, it may contain degenerate primers.

When detecting norovirus, which is one of the RNA viruses having no envelope in the present invention, as an example of a primer pair, as a primer for detecting norovirus, the notification of the monitoring and safety section of the Safety Department, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare (food safety). Examples thereof include the primers (SEQ ID NOs: 1 to 5) described in the primers described in the envelope No. 1105001), but the present invention is not limited thereto. In the primers described above, Norovirus G1 type is detected by SEQ ID NOs: 1 and 2, and Norovirus G2 type is detected by SEQ ID NOs: 3-5. The concentration of the primer to be detected is not particularly limited, but the concentration of the forward primer is 0.1 μM or more and 3 μM or less and the concentration of the reverse primer is 0.1 μM or more and 3 μM with respect to the entire RT-PCR reaction solution. The following is preferable. More preferably, the concentration of the forward primer is 0.1 μM or more and 2 μM or less, and the concentration of the reverse primer is 0.5 μM or more and 2 μM or less.

In another embodiment, when the present invention detects coronavirus (SARS-nCOV-2), which is one of the enveloped RNA viruses, an example of a primer pair is a "pathogen" published by the National Institute of Infectious Diseases. Sequences described in "Detection Manual 2019-nCoV" (SEQ ID NOs: 10, 11, 13, 14), "2019-Novel Coronavirus (2019-nCoV) Real-time RT-pCR Panel Primers and Probes" announced by the Centers for Disease Control and Prevention. (SEQ ID NOs: 16, 17, 19, 20, 22, 23) can be suitably used in the present invention, but the present invention is not limited thereto. In the primer sequences described above, the nucleocapsid protein (N) region of SARS-nCOV-2 is detected by SEQ ID NOs: 10 and 11, 13 and 14, 16 and 17, 19 and 20, 22 and 23. In the detection of coronaviruses such as SARS-nCOV-2, nucleocapsid (N) region, envelope protein (E) region, spike protein (S) region, RNA-dependent RNA polymerase (RdRp) region, Open Reading Frame. Genes such as (ORF) region can be detected, but the detection is not limited thereto. As for the concentration of the primer to be used, it is preferable that the concentration of the forward primer is 0.1 μM or more and 3 μM or less and the concentration of the reverse primer is 0.1 μM or more and 3 μM or less with respect to the entire RT-PCR reaction solution. .. More preferably, the concentration of the forward primer is 0.1 μM or more and 2 μM or less, and the concentration of the reverse primer is 0.5 μM or more and 2 μM or less.

One feature of the present invention is to detect two or more target nucleic acids by using two or more kinds of fluorescent compounds. Therefore, in the present invention, probes labeled with two or more kinds of fluorescent compounds can be used. In yet another embodiment, there is a detection method in which at least one type of labeled hybridization probe and a double-stranded DNA-bound fluorescent compound are combined to utilize two or more types of fluorescent compounds. By using two or more kinds of fluorescent compounds in this way, the analysis of the amplified product can be monitored by monitoring the fluorescent signal instead of the usual electrophoresis, and the analysis labor is reduced. Furthermore, it is not necessary to open the reaction vessel, and the risk of contamination can be reduced. It is also possible to identify each target nucleic acid separately by labeling each hybridization probe with a different fluorescent dye, corresponding to the type and subtype of virus and microorganism.

Examples of the double-stranded DNA-bound fluorescent compound include SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), and EvaGreen (registered trademark; Biotium). , LCGreen (Idaho), LightCycler (registered trademark) 480 ResoLight (Roche Applied Science) and the like.

Examples of the hybridization probe used in the present invention include TaqMan hydrolysis probe (US Pat. No. 5,210,015, US Pat. No. 5,538,848, US Pat. No. 5,487,972). , US Pat. No. 5,804,375), Molecular Beacon (US Pat. No. 5,118,801), FRET Hybridization Probe (International Publication No. 97/46707, International Publication No. 97/46712) , International Publication No. 97/46714 pamphlet) and the like.

As the fluorescent compound that can be used for the hybridization probe, any fluorescent compound known in the art can be used, and for example, it can be selected according to the qPCR device to be used. Specific examples of the fluorescent compound include, for example, rhodamine (ROX) or a derivative thereof (eg, 5-carboxy-X-rhodamine, 6-carboxy-X-rhodamine, 5-carboxyrhodamine 6G (CR6G), tetramethylrhodamine (TAMRA). )), Or rhodamine-based compounds such as salts thereof; fluorosane or derivatives thereof (eg, FAM (carboxyfluorescein), JOE (6-carboxy-4', 5'-dichloro 2', 7'-dimethoxyfluoresane),. FITC (fluorescein isothiocyanate), TET (tetrachlorofluorescein), HEX (5'-hexachloro-fluoresane-CE phosphoroamidite), VIC®, BODICY® series, rhodamine or derivatives thereof (eg,) Examples thereof include 5-carboxyrhodamine 6G (CR6G), tetramethylrhodamine (TAMRA)), Cy® dyes (eg, Cy3, Cy5), derivatives thereof, and non-rhodamine compounds such as salts thereof. However, it is not limited to these. As the fluorescent compound, a quenching substance suitable for the fluorescent substance to be used can be used, if necessary. Examples of the quenching substance corresponding to the above-mentioned fluorescent substance include TAMRA (tetramethyl-rhodamine), DABCYL (4- (4-dimethylaminophenylazo) benzoic acid), BHQ1 (BHQ: Black Hole Quencher (registered trademark)). )), BHQ2, BHQ3 and the like, but are not limited thereto.

In one embodiment, as the base sequence of the hybridization probe for detecting norovirus, the sequence (SEQ ID NO: 6-9) described in the notification (Food Safety Supervision No. 1105001) of the Monitoring and Safety Division, Safety Department, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labor and Welfare is used. It can be mentioned, but it is not limited to this. In the probe sequence described above, norovirus G1 type is detected by SEQ ID NO: 6 or 7, and norovirus G2 type is detected by SEQ ID NO: 8 or 9. In addition, if the target nucleic acid consists of subtypes, it may contain degenerate sequences. The concentration of the fluorescently labeled probe is preferably 0.01 μM or more and 1.0 μM or less. More preferably, it is 0.013 μM or more and 0.75 μM or less, and even more preferably 0.02 μM or more and 0.5 μM or less.

In another embodiment, when coronavirus coronavirus (SARS-nCOV-2), which is one of the enveloped RNA viruses, is detected in the present invention, the base sequence of the hybridization probe for the detection is as a national infectious disease study. The sequences (SEQ ID NOs: 12 and 15) described in the "Pathogen Detection Manual 2019-nCoV" published by the Institute and the "2019-Novel Coronavirus (2019-nCoV) Real-time RT-" published by the American Center for Disease Control and Prevention. "PCR Panel Primers and Probes" (SEQ ID NOs: 18 and 21 and 24) can be preferably used in the present invention, but the present invention is not limited thereto. The probe sequence described above detects the N region of SARS-nCOV-2. In addition, if the target nucleic acid consists of subtypes, it may contain degenerate sequences. In the detection of coronaviruses such as SARS-nCOV-2, genes such as N region, E region, S region, RdRp region, ORF region can be detected, but it is not particularly limited to this. do not have. The concentration of the fluorescently labeled probe is preferably 0.01 μM or more and 1.0 μM or less. More preferably, it is 0.013 μM or more and 0.75 μM or less, and even more preferably 0.02 μM or more and 0.5 μM or less.

Another aspect of the present invention is a test kit or composition for detecting nucleic acids derived from a virus or pathogenic microorganism in a sample, which is a visible dye used for preparing a sample mixture and contamination resistance. Multiplex PCR using two or more fluorescent compounds to test for pathogenic microorganisms in a sample that does not undergo nucleic acid isolation and may contain contaminants, characterized by containing a PCR reaction solution containing the DNA polymerase A testing kit or composition for doing by law.

The type and amount of visible dye used in this embodiment, the type and amount of DNA polymerase having contamination resistance, the type and amount of primer or probe, the type of sample used for inspection, pathogenic microorganisms to be inspected, etc. , The same as those detailed in the above inspection method.

Hereinafter, the present invention will be specifically described with reference to Examples. However, the present invention is not limited to the following examples.

Test example 1. Measurement of saliva turbidity

(1) Preparation of sample

SARS-CoV-2 coronavirus-negative saliva specimens were suspended in sterile water to 50 w / w%.

(2) Measurement of turbidity (OD660)

The OD660 of the saliva suspension was measured. The measurement result was multiplied by the dilution ratio to determine the turbidity of the collected saliva.

(3) Result

It was confirmed that the turbidity of the collected saliva sample was 1.03 Abs. Subsequent studies using the saliva sample used the saliva sample.

Test example 2. Examination of visibility of aqueous solution containing visible dye

(1) Preparation of visible dye

The following visible dyes were dissolved in sterile water at a concentration of 0.1 to 0.000000001 w / w% to prepare an aqueous solution.

Condition 1 Cresol Red

Condition 2 Orange G

Condition 3 Tartrazine

Condition 4 Patent Blue

Condition 5 Acid red 18 (Ponceau 4R)

Condition 6 Ponso S

Condition 7 Phenol Red

Condition 8 BPB (bromophenol blue)

Condition 9 Patent Blue and Tartrazine

Condition 10 No visible pigment (sterile water)

(2) Dispensing of visible dye aqueous solution and confirmation of visibility

50 μL of each visible dye aqueous solution was dispensed into the PCR reaction tube. After dispensing, the contents were checked from the top of the tube to see if the color tone could be visually recognized.

(3) Result

Although there was a difference in visibility depending on the color tone, it was confirmed that the color tone could be easily visually recognized up to a visible dye concentration of 0.00001 w / w%.

Test example 3. Confirmation of absorption wavelength of visible dye

(1) Preparation of visible dye

The following visible dyes were dissolved in sterile water at a concentration of 0.0001 w / w% or 0.00001 w / w% to prepare an aqueous solution.

Condition 1 Cresol Red

Condition 2 Orange G

Condition 3 Tartrazine

Condition 4 Patent Blue

Condition 5 Acid red 18

Condition 6 Ponso S

Condition 7 Phenol Red

Condition 8 BPB

Condition 9 Patent Blue + Tartrazine

(2) Absorbance measurement

The maximum absorption wavelength and absorbance of each visible dye aqueous solution were measured using an absorptiometer (optical path length 10 mm).

(3) Result

The results of measuring the absorbance at the visible concentration of each visible dye are as shown in Table 2.

Test Example 4.2 Effect of visible dye on fluorescence detection of multiplex RT-PCR in 1-step RT-PCR reaction of enzyme system (norovirus RNA detection)

(1) Preparation of norovirus RNA

Synthetic RNAs of G1 and G2 norovirus were mixed to 1250 copies / μL.

(2) Reaction solution

Norovirus in the reaction solution was detected in the two-enzyme system 1-step RT-PCR using the reaction solution having the composition shown below as the basic composition.

Reaction solution

(Norovirus detection kit G1 / G2-high-speed probe detection Quick Step- (Toyobo) attachment)

Primer solution (5 μL of 10x primer solution)

(Norovirus detection kit G1 / G2-high-speed probe detection Quick Step- (Toyobo) attachment)

Probe solution (5 μL of 10 x probe solution)

(Norovirus Detection Kit G1 / G2-Fast Probe Detection Quick Step-Attachment)

Pretreatment liquid 3 μL

(Norovirus detection kit G1 / G2-high-speed probe detection Quick Step- (Toyobo) attachment)

4.2 ng / μL rTth DNA polymerase (Toyobo)

0.01 μg / μL anti-Tth antibody

0.1U / μL MMLV R Tase (Toyobo)

Each of the above reagents was mixed to prepare an RT-PCR reaction solution so that the final liquid volume was 48 μL.

(3) Addition of visible dye

1 μL of the following visible dye adjusted so that the final PCR concentration is 0.001% to 0.00001% together with 1 μL of 1250 copies / μL of norovirus synthetic RNA is added to the reaction solution prepared in the previous step (2). The reaction system was 50 μL.

Condition 1 Cresol Red

Condition 2 Orange G

Condition 3 Tartrazine

Condition 4 Patent Blue

Condition 5 Acid red 18

Condition 6 Ponso S

Condition 7 Phenol Red

Condition 8 BPB

Condition 9 No visible pigment (sterile water)

(4) PCR reaction

The PCR reaction conditions were the optimum conditions described in Norovirus Detection Kit G1 / G2-Fast Probe Detection Quick Step- (Toyobo). As the measuring machine stand, CFX96WELL DEEP manufactured by BioRad was used. For the detection of each amplified product, a TaqMan (R) probe corresponding to G1 type norovirus (Cy5 channel), G2 type norovirus (ROX channel), and internal standard control (FAM channel) was used.

(5) Result

For the measurement results, the Ct value and the reached fluorescence intensity were calculated using BioRad's CFX Manager or CFX Maestro software with a threshold value of 50. The results are shown in FIGS. 1 and 3. Depending on the type of visible dye, a decrease in the reached fluorescence intensity was confirmed, but under all conditions, two types of norovirus and a total of three types of signals of the internal standard control were detected as positive.

 

試験例６．可視色素存在下における様々なＤＮＡポリメラーゼを利用したＳＡＲＳ－ＣｏＶ－２コロナウイルス検体のマルチプレックス検出検討（２酵素系１ステップＲＴ－ＰＣＲ反応）

（１－１）ＳＡＲＳ－ＣｏＶ－２コロナウイルス検体の調製

　ＳＡＲＳ－ＣｏＶ－２コロナウイルス検体であるＳＡＲＳ－ＣｏＶ－２　（ｒｅｃｏｍｂｉｎａｎｔ）　Ｓｔｏｃｋ（ＺｅｐｔｏＭｅｔｒｉｘ、ｉｎｔａｃｔ）を利用した。検体を２０コピー／μＬとなるように調製した。

（１－２）唾液懸濁液の採取

　ＳＡＲＳ－ＣｏＶ－２コロナウイルスの陰性が確認されている５００μＬ唾液検体を採取した。

（１－３）可視色素溶液の調製

　Ａｃｉｄ　ｒｅｄ　１８を０．０５％になるようにリン酸バッファー（ＰＢＳ（－））に溶解し可視色素溶液を調製した。

（１－４）ＳＡＲＳ－ＣｏＶ－２コロナウイルス検体および唾液の前処理

　可視色素溶液 １μＬ、ＳＡＲＳ－ＣｏＶ－２コロナウイルス検体２０コピー／μＬを１μＬ、唾液１μＬを混合した。検体を含む可視色素溶液３μＬに前処理液 ３μＬ（ＳＡＲＳ－ＣｏＶ－２　Ｄｅｔｅｃｔｉｏｎ　Ｋｉｔ　－Ｍｕｌｔｉ－（東洋紡）添付品）を混合し、９５℃５分間の熱処理を行った後に、前処理済検体液（可視色素を含む試料混合液に相当）とした。なお、ここで行った前処理法は核酸の単離処理を伴わない。

（２－１）反応液

　以下に示される組成の反応液を基本組成とし、２酵素系１ステップＲＴ－ＰＣＲにおいて、反応液中のＳＡＲＳ－ＣｏＶ－２コロナウイルスを検出した。

反応液

　（ＳＡＲＳ－ＣｏＶ－２　Ｄｅｔｅｃｔｉｏｎ　Ｋｉｔ　－Ｍｕｌｔｉ－（東洋紡）添付品）

４．２ｎｇ／μＬ　各ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ

０．０１μｇ／μＬ　各抗ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ　抗体

０．１Ｕ／μＬ ＭＭＬＶ　ＲＴａｓｅ（東洋紡）

　前記各試薬と、（２－２）に記載のプライマー・プローブ液を混合し、最終液量が４４μＬとなるようにＲＴ－ＰＣＲ反応液を調製した。これに前処理済検体液（可視色素を含む試料混合液に相当）６μＬ添加し、最終液量５０μＬとして工程（４）の条件にてＰＣＲを実施した。（可視色素のＰＣＲ反応液中濃度は０．００１％）

（２－２）プライマー・プローブ液

　プラーマおよびプローブは、アメリカ疾病予防管理センター（ＣＤＣ）発行「２０１９－Ｎｏｖｅｌ　Ｃｏｒｏｎａｖｉｒｕｓ　（２０１９－ｎＣｏＶ）　Ｒｅａｌ－ｔｉｍｅ　ＲＴ－ＰＣＲ　Ｐａｎｅｌ　Ｐｒｉｍｅｒｓ　ａｎｄ　Ｐｒｏｂｅｓ」（Ｅｆｆｅｃｔｉｖｅ：　２４　Ｊａｎ　２０２０）　に記載されているＮ１およびＮ２セットの配列であり、各増幅物産物の検出には、Ｎ１（Ｃｙ５チャネル）、Ｎ２（ＲＯＸチャネル）に対応するＴａｑＭａｎ（Ｒ）プローブを利用した。ＲＴ－ＰＣＲ反応液中のプローブおよびプライマーの濃度は同文献に記載の濃度を添加した。

（３）使用したＤＮＡポリメラーゼおよびその変異体

　以下に示されるＤＮＡポリメラーゼおよびその変異体を、記載の濃度にてそれぞれ反応に使用した。変異体に関する表記は、アミノ酸の1文字略語表記に従う。変異導入部位に関しては酵素の名称に含む数字となっており、左に変更前のアミノ酸、右に変更後のアミノ酸を記載した。例えば、「Ｔｔｈ変異体（Ｍ７４９Ｋ）」は、Ｔｔｈ　ＤＮＡ　ｐｏｌｙｍｅｒａｓｅの７４９位のＭ（メチオニン）をＫ（リシン）に変異させていることを意味する。

（使用酵素）

・酵素１：Ｔａｑ　ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ（野生型）（東洋紡）

・酵素２：Ｔｔｈ　ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ（野生型）（東洋紡）

・酵素３：Ｔｔｈ変異体（Ｑ５０９Ｒ）

・酵素４：Ｔｔｈ変異体（Ｑ５０９Ｋ）

・酵素５：Ｔｔｈ変異体（Ｅ６２８Ｋ）

・酵素６：Ｔｔｈ変異体（Ｍ７４９Ｋ）

・酵素７：Ｔｔｈ変異体（Ｆ７５１Ｙ）

・酵素８：Ｔｔｈ変異体（Ｄ５４９Ｇ）

・酵素９：ＨａｗｋＺ０５ ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ（Ｒｏｃｈｅ）

（４）ＰＣＲ反応

ＰＣＲ反応条件は、ＳＡＲＳ－ＣｏＶ－２　Ｄｅｔｅｃｔｉｏｎ　Ｋｉｔ　－Ｍｕｌｔｉ－（東洋紡）に記載の至適条件にて行った。測定機台はＢｉｏＲａｄ製ＣＦＸ９６ＷＥＬＬ　ＤＥＥＰを使用した。

（５）結果

　測定結果は、ＢｉｏＲａｄ製ＣＦＸ ＭａｎａｇｅｒまたはＣＦＸ Ｍａｅｓｔｒｏソフトウエアにて、閾値を１００としてＣｔ値および到達蛍光強度を算出した。この結果を図３及び表５に示す。Ｔｔｈ ＤＮＡポリメラーゼおよびその変異体と、ＨａｗｋＺ０５ ＤＮＡポリメラーゼでは、検体添加時においてもＳＡＲＳ－ＣｏＶ－２コロナウイルスＮ１領域およびＮ２領域のすべてにおいて陽性として検出された。反面、ＴａｑＤＮＡポリメラーゼではすべて陰性となった。

Test example 5. Multiplex detection study of SARS-CoV-2 coronavirus sample using TthDNA polymerase in the presence of saliva sample and visible dye (2-enzyme system 1-step RT-PCR reaction)

(1-1) Preparation of SARS-CoV-2 coronavirus sample

A SARS-CoV-2 coronavirus sample, SARS-CoV-2 (recombinant) Stock (ZeptoMetrix, intact), was used. The sample was prepared to be 20 copies / μL.

(1-2) Collection of saliva suspension

A 500 μL saliva sample confirmed to be negative for SARS-CoV-2 coronavirus was collected.

(1-3) Preparation of visible dye solution

The following visible dye was dissolved in a phosphate buffer (PBS (−)) so as to have a concentration of 0.05% to prepare a visible dye solution.

Condition 1 No visible pigment (sterile water)

Condition 2 Acid red 18

Condition 3 Tartrazine

Condition 4 Patent Blue

Condition 5 BPB

(1-4) Pretreatment of SARS-CoV-2 coronavirus sample and saliva 1 μL of visible dye solution, 20 copies / μL of SARS-CoV-2 coronavirus sample / μL were mixed, and 1 μL of saliva was mixed. 3 μL of the visible dye solution containing the sample is mixed with 3 μL of the pretreatment liquid (SARS-CoV-2 Detection Kit-Multi- (Toyobo) attachment), heat-treated at 95 ° C. for 5 minutes, and then the pretreated sample liquid ( Equivalent to a sample mixture containing a visible dye). The pretreatment method performed here does not involve the nucleic acid isolation treatment.

(2-1) Reaction solution

Using the reaction solution having the composition shown below as the basic composition, SARS-CoV-2 coronavirus in the reaction solution was detected by 2-enzyme system 1-step RT-PCR.

Reaction solution

(SARS-CoV-2 Detection Kit -Multi- (Toyobo) attachment)

4.2 ng / μl rTth DNA polymerase (Toyobo)

0.01 μg / μL anti-Tth antibody

0.1U / μL MMLV R Tase (Toyobo)

Each of the above reagents was mixed with the primer / probe solution described in (2-2) to prepare an RT-PCR reaction solution so that the amount of the solution was 44 μL. 6 μL of the pretreated sample solution (corresponding to the sample mixture containing the visible dye) was added thereto, and PCR was carried out under the conditions of step (4) with a final liquid volume of 50 μL. (Concentration of visible dye in PCR reaction solution is 0.001%)

(2-2) Primer / probe solution

Plumers and probes are described in "2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effective: 24 Jan 2020), published by the Centers for Disease Control and Prevention (CDC), N1 and N2. A TaqMan (R) probe corresponding to N1 (Cy5 channel) and N2 type (ROX channel) was used to detect each amplification product, which is a set sequence. As the concentration of the probe and the primer in the RT-PCR reaction solution, the concentration described in the same document was added.

(4) PCR reaction

The PCR reaction conditions were the optimum conditions described in SARS-CoV-2 Detection Kit-Multi- (Toyobo). As the measuring machine stand, CFX96WELL DEEP manufactured by BioRad was used. Other conditions are the same as in Example 1 above.

(5) Result

For the measurement results, the Ct value and the reached fluorescence intensity were calculated using BioRad's CFX Manager or CFX Maestro software with the threshold value set to 100. The results are shown in FIGS. 2 and 4. Detection of N1 and N2 regions was confirmed under all conditions, even in the presence of saliva.

Test Example 6. Multiplex detection study of SARS-CoV-2 coronavirus sample using various DNA polymerases in the presence of visible dye (2-enzyme system 1-step RT-PCR reaction)

(1-1) Preparation of SARS-CoV-2 coronavirus sample

A SARS-CoV-2 coronavirus sample, SARS-CoV-2 (recombinant) Stock (ZeptoMetrix, intact), was used. The sample was prepared to be 20 copies / μL.

(1-2) Collection of saliva suspension

A 500 μL saliva sample confirmed to be negative for SARS-CoV-2 coronavirus was collected.

(1-3) Preparation of visible dye solution

A visible dye solution was prepared by dissolving Acid red 18 in a phosphate buffer (PBS (−)) so as to have a concentration of 0.05%.

(1-4) Pretreatment of SARS-CoV-2 coronavirus sample and saliva

1 μL of visible dye solution, 1 μL of SARS-CoV-2 coronavirus sample 20 copies / μL, and 1 μL of saliva were mixed. 3 μL of the visible dye solution containing the sample is mixed with 3 μL of the pretreatment liquid (SARS-CoV-2 Detection Kit-Multi- (Toyobo) attachment), heat-treated at 95 ° C. for 5 minutes, and then the pretreated sample liquid ( Equivalent to a sample mixture containing a visible dye). The pretreatment method performed here does not involve the nucleic acid isolation treatment.

(2-1) Reaction solution

Using the reaction solution having the composition shown below as the basic composition, SARS-CoV-2 coronavirus in the reaction solution was detected by 2-enzyme system 1-step RT-PCR.

Reaction solution

(SARS-CoV-2 Detection Kit -Multi- (Toyobo) attachment)

4.2 ng / μL Each DNA polymerase

0.01 μg / μL Each anti-DNA polymerase antibody

0.1U / μL MMLV R Tase (Toyobo)

Each of the above reagents was mixed with the primer / probe solution described in (2-2) to prepare an RT-PCR reaction solution so that the final volume was 44 μL. 6 μL of the pretreated sample solution (corresponding to the sample mixture containing the visible dye) was added thereto, and PCR was carried out under the conditions of step (4) with a final liquid volume of 50 μL. (Concentration of visible dye in PCR reaction solution is 0.001%)

(2-2) Primer / probe solution

Plumers and probes are described in "2019-Novel Coronavirus (2019-nCoV) Real-time RT-PCR Panel Primers and Probes" (Effective: 24 Jan 2020), published by the Centers for Disease Control and Prevention (CDC), N1 and N2. A TaqMan (R) probe corresponding to N1 (Cy5 channel) and N2 (ROX channel) was used to detect each amplification product, which is a set sequence. As the concentration of the probe and the primer in the RT-PCR reaction solution, the concentration described in the same document was added.

(3) DNA polymerase used and its variants

The DNA polymerases and variants thereof shown below were used in the reactions at the concentrations listed below. The notation for variants follows the one-letter abbreviation notation for amino acids. Regarding the mutation introduction site, the numbers are included in the name of the enzyme, and the amino acid before the change is shown on the left and the amino acid after the change is shown on the right. For example, "Tth mutant (M749K)" means that M (methionine) at position 749 of Tth DNA polymerase is mutated to K (lysine).

(Enzyme used)

・ Enzyme 1: Taq DNA polymerase (wild type) (Toyobo)

・ Enzyme 2: Tth DNA polymerase (wild type) (Toyobo)

-Enzyme 3: Tth mutant (Q509R)

-Enzyme 4: Tth mutant (Q509K)

Enzyme 5: Tth mutant (E628K)

Enzyme 6: Tth mutant (M749K)

Enzyme 7: Tth mutant (F751Y)

Enzyme 8: Tth mutant (D549G)

Enzyme 9: HawkZ05 DNA polymerase (Roche)

(4) PCR reaction

The PCR reaction conditions were the optimum conditions described in SARS-CoV-2 Detection Kit-Multi- (Toyobo). As the measuring machine stand, CFX96WELL DEEP manufactured by BioRad was used.

(5) Result

For the measurement results, the Ct value and the reached fluorescence intensity were calculated using BioRad's CFX Manager or CFX Maestro software with the threshold value set to 100. The results are shown in FIGS. 3 and 5. The Tth DNA polymerase and its variants and the HawkZ05 DNA polymerase were detected as positive in all of the SARS-CoV-2 coronavirus N1 and N2 regions even when the sample was added. On the other hand, all were negative with TaqDNA polymerase.

INDUSTRIAL APPLICABILITY The present invention is suitably used in molecular biology research, clinical tests, food hygiene control, tests for the purpose of preventing the spread of infectious diseases, and the like.

Claims (50)

A method for inspecting the presence or absence of two or more target nucleic acids in a sample with one PCR reaction solution, which comprises the following steps:

(1) A step of preparing a sample mixture containing a sample that has not been isolated from nucleic acid and a visible dye.

(2) A step of mixing the sample mixture prepared in the above step (1) with a PCR reaction solution containing a DNA polymerase having contamination resistance.

(3) A step of carrying out a PCR reaction after sealing the reaction vessel; and

(4) A step of detecting two or more target nucleic acids with two or more types of fluorescent compounds.

The method according to claim 1, wherein the DNA polymerase having contamination resistance is a DNA polymerase having reverse transcription activity.

The method according to claim 1, wherein the PCR reaction solution further contains reverse transcriptase.

Any of claims 1 to 3, wherein the sample is at least one selected from the group consisting of a saliva sample, a sputum sample, a mouthwash, a tear fluid, a pharyngeal swab sample, a nasal swab sample, a fecal sample, and a wiping test sample. The method described in Crab.

Claims 1 to 1, wherein the sample is a suspension suspended in at least one selected from the group consisting of water, physiological saline, a buffer solution, and a sputazyme enzyme solution, or a centrifugal supernatant or a concentrate thereof. The method according to any one of 4.

The method according to any one of claims 1 to 5, wherein the DNA polymerase having contamination resistance is a DNA polymerase belonging to Family A.

Any of claims 1 to 6, wherein the DNA polymerase having contamination resistance is at least one DNA polymerase having contamination resistance selected from the group consisting of Tth, Hawk Z05 and variants thereof. The method described in Crab.

The variant consists of an amino acid sequence showing 90% or more identity with the amino acid sequence of Tth polymerase (SEQ ID NO: 25) or Hawk Z05 polymerase (SEQ ID NO: 26), and exhibits contamination-resistant DNA polymerase activity. The method according to claim 7, which is a thing.

The variant consists of an amino acid sequence having one or several amino acid deletions, substitutions and / or additions in the amino acid sequence of Tth polymerase (SEQ ID NO: 25) or Hawk Z05 polymerase (SEQ ID NO: 26) and is contaminated. The method according to claim 7 or 8, wherein the DNA polymerase activity is resistant to substances.

Claims 3-9, wherein the reverse transcriptase is a reverse transcriptase derived from at least one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and variants thereof. The method described in any of.

Visible dyes are purple (400-420 nm), indigo color (420-440 nm), blue (440-490 nm), green (490-570 nm), yellow (570-585 nm), orange (585-620 nm), red (620). The method according to any one of claims 1 to 10, wherein the dye exhibits a color tone (maximum absorption wavelength in parentheses) selected from the group consisting of (-780 nm), white, black, and gray.

Visible pigments are amaranth, erythrosin, alla red, ponceau, phenol red, orange G, ponso S, cresol red, rose bengal, tartrazine, sunset yellow, fast green, bromocresol green, brilliant blue, indigo carmine, patent blue. The method according to any one of claims 1 to 11, which is at least one selected from the group consisting of, bromophenol blue, and bromocresol purp.

Claimed using a visible dye-containing solution in which the visible dye is suspended in at least one solvent selected from the group consisting of water, physiological saline, a buffer solution, an acidic solution, an alkaline solution, and an organic solvent. The method according to any one of 1 to 12.

The method according to any one of claims 1 to 13, wherein the concentration of the visible dye in the visible dye-containing solution is 0.00001% or more.

The method according to any one of claims 1 to 14, wherein the concentration of the visible dye in the PCR reaction solution is 0.000001% or more.

The method according to any one of claims 1 to 15, wherein at least one of the fluorescent compounds is a double-stranded DNA-bound fluorescent compound.

Double-stranded DNA-bound fluorescent compounds are SYBR® Green I, SYBR® Gold, SYTO-9, SYTP-13, SYTO-82, EvaGreen®, LCGreen, and LightCycler®. Trademark) The method according to claim 16, wherein the method is one or more selected from the group consisting of 480 ResoLight.

The method according to any one of claims 1 to 17, wherein at least one of the fluorescent compounds is a hybridization probe.

18. The method of claim 18, wherein the hybridization probe is a TaqMan probe.

The method according to any one of claims 1 to 19, wherein the two or more kinds of fluorescent compounds include a combination of a hybridization probe and a double-stranded DNA-bound fluorescent compound.

The method according to any one of claims 1 to 20, wherein at least one of the two or more target nucleic acids in the sample is a nucleic acid derived from a pathogenic microorganism.

21. The method of claim 21, wherein the pathogenic microorganism is a DNA virus or RNA virus.

22. The method of claim 22, wherein the RNA virus is an enveloped RNA virus.

An enveloped RNA virus is selected from the group consisting of flavivirus family virus; togavirus family virus; coronavirus family virus; orthomixovirus family virus; bunyavirus family virus; paramyxovirus family virus; and phyllovirus family virus. The method according to claim 23, which is at least one type.

The method according to claim 23 or 24, wherein the RNA virus having an envelope is a coronaviridae virus.

Claim 24, wherein the coronavirus family virus is at least one selected from the group consisting of SARS (Severe Acute Respiratory Syndrome) coronavirus, MERS (Middle East Respiratory Syndrome) coronavirus, and SARS-nCOV-2 coronavirus. Or the method according to 25.

22. The method of claim 22, wherein the RNA virus is an RNA virus without an envelope.

Claimed that the non-enveloped RNA virus is at least one selected from the group consisting of astroviridae virus; caliciviridae virus; picornaviridae virus; hepeviridae virus; and leoviridae virus. 27.

28. The method of claim 27 or 28, wherein the non-enveloped RNA virus is norovirus or rotavirus.

The method according to any one of claims 27 to 29, wherein the RNA virus having no envelope is a norovirus, and it is possible to determine whether the norovirus is a GI type or a GII type.

The method according to any one of claims 1 to 30, wherein the PCR reaction solution further comprises at least one selected from the group consisting of bovine serum albumin and gelatin.

The method according to any one of claims 1 to 31, wherein the PCR reaction solution further contains 1 mM or more of divalent cations.

Two or more types of samples not subjected to nucleic acid isolation treatment, which comprises a visible dye used for preparing a sample mixture and a PCR reaction solution containing a DNA polymerase having resistance to contaminants. A test kit or composition used to test for the presence or absence of two or more target nucleic acids in a fluorescent compound of.

The kit or composition according to claim 33, wherein the DNA polymerase having contamination resistance is a DNA polymerase having reverse transcription activity.

The kit or composition according to claim 33, wherein the PCR reaction solution further comprises reverse transcriptase.

The kit or composition according to any one of claims 33 to 35, wherein the DNA polymerase having contamination resistance is a DNA polymerase belonging to Family A.
13. The kit or composition described in the above.

The variant consists of an amino acid sequence showing 90% or more identity with the amino acid sequence of Tth polymerase (SEQ ID NO: 25) or Hawk Z05 polymerase (SEQ ID NO: 26), and exhibits contamination-resistant DNA polymerase activity. The kit or composition according to claim 37.

The variant consists of an amino acid sequence having one or several amino acid deletions, substitutions and / or additions in the amino acid sequence of Tth polymerase (SEQ ID NO: 25) or Hawk Z05 polymerase (SEQ ID NO: 26) and is contaminated. The kit or composition according to claim 37 or 38, which exhibits DNA polymerase activity having resistance to substances.

Claims 35-39, wherein the reverse transcriptase is a reverse transcriptase derived from at least one selected from the group consisting of Moloney murine leukemia virus (MMRV), avian myeloblastosis virus (AMV) and variants thereof. The kit or composition according to any of the above.

Visible dyes are purple (400-420 nm), indigo (420-440 nm), blue (440-490 nm), green (490-570 nm), yellow (570-585 nm), orange (585-620 nm), red (620). The kit or composition according to any one of claims 33 to 40, which is a dye exhibiting a color tone (maximum absorption wavelength in parentheses) selected from the group consisting of (-780 nm), white, black, and gray.

Visible pigments are Amaranth, Erythrosine, Arla Red, Ponceau, Phenol Red, Orange G, Ponso S, Cresol Red, Rose Bengal, Tartrazine, Sunset Yellow, Fast Green, Bromophenol Green, Brilliant Blue, Indigo Carmine, Patent Blue. The kit or composition according to any one of claims 33 to 41, which is at least one selected from the group consisting of, bromophenol blue, and bromocresol purp.

The visible dye comprises a visible dye-containing solution in which the visible dye is suspended in at least one solvent selected from the group consisting of water, physiological saline, a buffer solution, an acidic solution, an alkaline solution, and an organic solvent. The kit or composition according to any one of 33 to 42.

The kit or composition according to any one of claims 33 to 43, wherein the concentration of the visible dye in the visible dye-containing solution is 0.00001% or more.

The kit or composition according to any one of claims 33 to 44, wherein the final concentration of the visible dye in the PCR reaction solution at the time of the PCR reaction is adjusted to 0.000001% or more.

The kit or composition according to any one of claims 33 to 45, wherein at least one of the fluorescent compounds is a double-stranded DNA-bound fluorescent compound.

Double-stranded DNA-bound fluorescent compounds are SYBR® Green I, SYBR® Gold, SYTO-9, SYTP-13, SYTO-82, EvaGreen®, LCGreen, and LightCycler®. The kit or composition according to claim 46, which is one or more selected from the group consisting of 480 ResoLight.

The kit or composition according to any one of claims 33 to 47, wherein at least one of the fluorescent compounds is a hybridization probe.

The kit or composition according to claim 48, wherein the hybridization probe is a TaqMan probe.

The kit or composition according to any one of claims 33 to 49, wherein the two or more fluorescent compounds include a combination of a hybridization probe and a double-stranded DNA-bound fluorescent compound.

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