WO2022039228A1

WO2022039228A1 - Improved multiplex pcr testing method

Info

Publication number: WO2022039228A1
Application number: PCT/JP2021/030390
Authority: WO
Inventors: 謙太寺内
Original assignee: 東洋紡株式会社
Priority date: 2020-08-21
Filing date: 2021-08-19
Publication date: 2022-02-24
Also published as: JPWO2022039228A1

Abstract

The purpose of the present invention is to provide an improved multiplex PCR testing method that has excellent workability, and that can simply detect multiple target nucleic acids with high sensitivity. This method for testing for the presence or absence of two or more target nucleic acids in a sample by using one PCR reaction solution is characterized by comprising the following steps: (1) a step for mixing a sample, which has not been subjected to a nucleic acid isolation process, with a PCR reaction solution that comprises a visible pigment and a DNA polymerase having contaminant resistance; (2) a step for carrying out a PCR reaction after sealing a reaction vessel; and (3) a step for detecting two or more target nucleic acids by using two or more types of fluorescent compound.

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, it is derived from a pathogenic microorganism by adding a reaction solution of a real-time polymerase chain reaction or a real-time reverse transcription polymerase chain reaction colored with a visible dye to a sample without isolating and purifying the nucleic acid in advance from the sample. Regarding the detection of nucleic acids. The method of the present invention can detect pathogenic microorganism-derived nucleic acids in, for example, fecal samples, blood samples, pharyngeal and nasal swab samples, saliva and sputum 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. Norovirus, which is one of the pathogenic RNA viruses, is a typical example of virus testing. Norovirus is a single-strand RNA virus that causes acute gastroenteritis. It is a virus of great public health concern because it is highly infectious and causes mass food poisoning and mass infection. In the pathogen test of norovirus, a tissue culture method has not been established, and a method for detecting a viral gene using electron microscopy, an immunological antigen detection method by ELISA, or a nucleic acid amplification technique has been developed. Of these, in Japan, the RT-PCR method based on the notification (Food Safety Supervision No. 1105001) of the Safety Department, Safety Department, Ministry of Health, Labor and Welfare has become widespread as an official method, and many tests for norovirus have been conducted. There is.

Norovirus is widely known to be classified by the genotypes of GI type norovirus and GII type norovirus. Then, it is desired to discriminate between GI type norovirus and GII type norovirus from the viewpoint of collecting epidemiological data such as estimation of infection route. Further, in order to reduce the number of tests, it is desirable to use multiplex RT-PCR in which two genotypes are simultaneously distinguished and detected in one measurement.

Conventionally, for detection of norovirus from stool samples, for example, a 10% suspension of stool is prepared, RNA is extracted and purified from the centrifugal supernatant using a commercially available viral RNA extraction kit, and this RNA extract is used. Norovirus has been detected (Shokuankan 1105001). However, in order to inspect a large number of samples in a short time, this RNA extraction work is complicated, and simplification of the work has been required.

In recent years, paying attention to this RNA extraction work, a method for efficiently detecting the presence or absence of a virus by exposing the viral RNA by simply adding a fecal sample to the pretreatment solution and subjecting the treatment solution to RT-PCR (Patent Documents). 1. There is also known a method for detecting a virus in which a mixed solution obtained by adding a fecal suspension to an RT-PCR reaction solution is directly heated and then RT-PCR is carried out (Patent Document 2). By using these methods, it is possible to omit the RNA extraction work and simplify the work process.

By the way, the work of dispensing the reaction solution of PCR 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. When the continuous dispensing work into the reaction vessel is carried out, mistakes such as omission of dispensing into the reaction vessel and multiple dispensing may occur. 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.

Generally, the reaction solution of PCR is colorless and transparent, and the amount of the reaction solution used is relatively small, so it is difficult to determine whether or not the reaction solution is dispensed into the reaction vessel and the amount of the dispensed solution. This point can be a problem because it is easy to overlook, especially when inspecting a large number of samples. Therefore, in recent years, PCR reaction solutions and addition buffers containing visible dyes have been developed to improve visibility during dispensing, and a method for easily confirming the presence or absence of dispensing and the amount of dispensing after dispensing is adopted. In some cases. 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 norovirus stool test, by omitting RNA extraction, PCR reaction inhibitors such as polysaccharides and insoluble substances contained in the stool sample are brought in. Is done. If such an inhibitor or an insoluble substance is present, the intensity such as the detected fluorescence intensity may decrease in the detection using the fluorescent compound, and the detection sensitivity may decrease accordingly.

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.

WO2019 / 017452 Pamphlet WO 2019/212023 Pamphlet Japanese Unexamined Patent Publication No. 2012-2439

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 studies and found that a sample that has not been isolated from nucleic acid and may contain contaminating substances and / or insoluble substances contains a specific DNA polymerase together with a visible dye. It is possible to detect two or more target nucleic acids (for example, target nucleic acids derived from pathogenic microorganisms such as viruses and bacteria) by subjecting them to a PCR reaction or an RT-PCR reaction after mixing with the PCR reaction solution. I found it.

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, the nucleic acid is added to the PCR reaction solution containing the visible dye or the RT-PCR reaction solution without performing the work of isolating and purifying the nucleic acid from the sample which may contain contaminants in advance, and the multiplex PCR reaction or the multiplex RT- We have found that nucleic acids derived from a plurality of pathogenic microorganisms can be detected only by performing a 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 mixing a sample that has not been isolated from nucleic acid and a PCR reaction solution containing a DNA polymerase having visible dye and contaminant resistance.

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

(3) 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, wherein the sample is at least one selected from the group consisting of a fecal sample, a saliva sample, a sputum sample, a mouthwash, a tear fluid, a pharyngeal swab sample, a nasal swab sample, and a 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 variant comprises 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 has 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, erythrosine, 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] The method according to any one of Items 1 to 12, wherein the concentration of the visible dye in the PCR reaction solution is 0.000001% or more.

[Item 14] The method according to any one of Items 1 to 13, wherein the fluorescent compound is a double-stranded DNA-bound fluorescent compound.

[Item 15] Double-stranded DNA-bound fluorescent compounds are SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82, EvaGreen (registered trademark; Biotium), LCGreen, Item 14. The method according to Item 14, wherein the method is one or more selected from the group consisting of Light Cycler (registered trademark) 480 ResoLight.

Item 16. The method according to any one of Items 1 to 15, wherein the fluorescent compound is a hybridization probe.

Item 17. The method according to Item 16, wherein the hybridization probe is a TaqMan probe.

[Item 18] The method according to any one of Items 1 to 17, wherein the two or more kinds of fluorescent compounds are a combination of a hybridization probe and a double-stranded DNA-bound fluorescent compound.

[Item 19] The method according to any one of Items 1 to 18, 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 20. The method according to Item 19, wherein the pathogenic microorganism is a pathogenic bacterium that causes food poisoning.

[Item 21] The method according to Item 20, wherein the pathogen causing food poisoning is at least one selected from the group consisting of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella.

[Item 22] The method according to Item 19, 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] Two or more targets with two or more fluorescent compounds in a sample not subjected to nucleic acid isolation treatment, which comprises a PCR reaction solution containing a visible dye and a DNA polymerase having contamination resistance. A test kit or composition used to test for the presence of nucleic acids.

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 variant comprises 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 has 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 33 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] The kit or composition according to any one of Items 33 to 42, 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 44] The kit or composition according to any one of Items 33 to 13, wherein the fluorescent compound is a double-stranded DNA-bound fluorescent compound.

[Item 45] The double-stranded DNA-bound fluorescent compound is SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82, EvaGreen (registered trademark; Biotium), LCGreen, Item 44. The kit or composition according to Item 44, which is one or more selected from the group consisting of Light Cycler (registered trademark) 480 ResoLight.

46. The method of any of Items 33-45, wherein the fluorescent compound is a hybridization probe.

47. The kit or composition according to Item 46, wherein the hybridization probe is a TaqMan probe.

[Item 48] The kit or composition according to any one of Items 33 to 47, wherein the two or more kinds of fluorescent compounds are a combination of a hybridization probe and a double-stranded DNA-bound fluorescent compound.

According to the present invention, a sample that has not isolated the nucleic acid of a microorganism and may contain a contaminating substance or an insoluble substance is added to a reaction solution containing a visible dye, and then a PCR reaction or an RT-PCR reaction is performed. Even when detecting the target nucleic acid, it is possible to inspect with sufficient sensitivity. By using the PCR reaction solution containing the visible dye, it is possible to suppress the dispensing error in the dispensing process of the reaction solution, so that the genetic test and the pathogenic microorganism test work are further streamlined. 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.

It is a figure which shows the example of the multiplex detection of the genomic DNA derived from a microorganism in the presence of various visible dyes. It is a figure which shows the example of the multiplex detection of the genomic DNA derived from a microorganism in the presence of a fecal sample and various visible dyes. It is a figure which shows the example of the multiplex detection of the genomic DNA derived from a microorganism in the presence of a fecal sample and various visible dyes using Taq DNA polymerase. It is a figure which shows the example of the multiplex detection (two enzyme system 1 step RT-PCR) of norovirus RNA in the presence of various visible dyes. It is a figure which shows the example of the multiplex detection (two enzyme system 1 step RT-PCR) of the norovirus sample in the presence of a fecal sample and 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 (1 enzyme system 1 step RT-PCR) of a fecal sample and a norovirus sample in the presence of various visible dyes. It is a figure which shows the example of the multiplex detection (two enzyme system 1 step RT-PCR) of the fecal sample using various DNA polymerases, and the norovirus sample in the presence of a visible dye. 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. It is a figure which shows the example of the multiplex detection (1 enzyme system 1 step RT-PCR) of the fecal sample using various DNA polymerases, and the norovirus 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 the 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. Two or more different target nucleic acids derived from pathogenic microorganisms are added to a sample containing contaminants by adding a PCR reaction solution (including RT-PCR reaction solution) containing a visible dye and a DNA polymerase having contamination resistance. It is a method for inspecting the presence of pathogenic microorganisms including detection by the above-mentioned 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 mixing a sample not subjected to nucleic acid isolation treatment with a PCR reaction solution containing a DNA polymerase having visible dye and contaminant resistance;

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

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

Here, in the step (1), the PCR reaction solution may be an RT-PCR reaction solution that also performs a reverse transcription reaction 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 (1) to (3) 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 (1) to (3). Furthermore, in step (2), preferably in both steps (2) and (3), it is preferable not to open or close the reaction vessel lid 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 suspension suspended in water or a buffer solution in advance, and a solid sample such as a fecal sample may be subjected to a PCR reaction. It may be added directly to the liquid.

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 pathogenic bacteria 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. The simple inspection method of the present invention is particularly 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 2), 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 tests 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 they are found to be 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 particularly 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 3, Non-Patent Document 4, Patent Document 3).

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 4, Non-Patent Document 5). 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 6). 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 tests including asymptomatic persons. There is concern that the burden on people will increase. In the continuous dispensing work in the inspection of such a large number of samples, mistakes such as dispensing omission and multiple dispensing may occur. Since these mistakes cause re-inspection and the like, it is strongly desired to develop an inspection method with improved workability. 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. However, 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 fecal samples. It is useful for detecting saliva sample, sputum sample, mouthwash, tear fluid, pharyngeal swab sample, and nasal swab sample. 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.

In the step (1), in order to facilitate the extraction of RNA or DNA from a robust structure such as a capsid or a cell wall, the sample is mixed with a buffer solution, an acid or alkaline solution, a solution containing an organic solvent, or the like. It's okay to have it. The acidic solution contained in the 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.

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 kitchen utensils such as cutting boards, kitchen knives, towels, and tableware, refrigerator handles and toilets, bathroom door knobs, washrooms, kitchens, toilets, bathroom faucets, cookers' hands and fingers, and bathrooms. , Toilets, washbasins, handrails, living rooms 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 the high turbidity test sample is mixed with a PCR reaction solution containing a visible dye and the color tone is further changed, two or more types of fluorescent compounds can detect two or more target nucleic acids. It may be possible to do.

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 dispensing the reaction solution of PCR 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. When the continuous dispensing work into the reaction vessel is carried out, mistakes such as omission of dispensing into the reaction vessel and multiple dispensing may occur. 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, the improvement of the dispensing work efficiency by containing the visible dye in the PCR reaction solution has more significance than the improvement of the work efficiency.

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 contained in the solution used in the present invention is not particularly limited, but is 0.1% w / w or less, preferably 0.01% w / w or less, more preferably 0.01% w / w or less, based on the entire PCR reaction solution. It is 0.001% w / w or less, more preferably 0.0001% w / w or less, and even more preferably 0.00001% 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 PCR reaction solution. 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 PCR cycle in step (2) 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 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.

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 (2), the PCR solution added to the mixture contains a DNA polymerase and, if necessary, a 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 (Mloney 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 transcription activity (for example, RTX: reverse transcription xenopolymerase), and the present invention has such reverse transcriptase activity. 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 a specific embodiment, 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 the effect of the present invention is exhibited, 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 an internal control or 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 the (ORF) region can be detected, but the detection is not limited thereto. As 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 acid derived from a virus or pathogenic microorganism in a sample, which comprises a PCR reaction solution containing a visible dye and a DNA polymerase having contamination resistance. A test kit or composition for testing for pathogenic microorganisms by multiplex PCR with two or more fluorescent compounds in a sample that does not contain nucleic acid isolation and may contain contaminants. It is a thing.

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 the test, the pathogenic microorganism to be tested, 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. Preparation of fecal suspension

(1) Preparation of sample

Norovirus-negative human fecal specimens were suspended in sterile water to 50 w / w%. This suspension was diluted 200-fold with water.

(2) Measurement of turbidity (OD660)

The OD660 of the stool suspension diluted 200-fold was measured. The measurement result was multiplied by the dilution ratio to determine the turbidity of the prepared fecal suspension.

(3) Result

It was confirmed that the turbidity of the prepared stool suspension was 41.8 Abs. Subsequent studies using the stool suspension used the stool suspension.

Test example 2. 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 3. 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 4. 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 (Ponceau 4R)

Condition 6 Ponso S

Condition 7 Phenol Red

Condition 8 BPB (bromophenol blue)

Condition 9 Patent Blue and 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 5. Examination of multiplex detection of microbial-derived genomic DNA using TthDNA polymerase in the presence of visible dye

(1) Preparation of genomic DNA derived from microorganisms

As a sample of the genome derived from microorganisms, the DNA of each food poisoning-causing bacterium was detected. Salmonella, Enterohemorrhagic Escherichia coli (Vircell), and Shigella (Zeptomatrix) were mixed and used at 1000 copies / μL.

(2) Reaction solution

PCR was performed using the reaction solution having the composition shown below as a basic composition and four types of templates: Salmonella, enterohemorrhagic Escherichia coli, Shigella, and internal standard control.

Reaction solution

(Gut microbiota gene detection kit-probe detection- (Toyobo) attachment)

Primer solution (2 μL of 10x primer solution)

(Gut microbiota gene detection kit-probe detection- (Toyobo) attachment)

Probe solution (2 μL of 10 x probe solution)

(Gut microbiota gene detection kit-probe detection- (Toyobo) attachment)

4.2 ng / μl rTth DNA polymerase (Toyobo)

0.01 μg / μl anti-Tth antibody

Each of the above reagents was mixed to prepare a PCR reaction solution so that the final liquid volume was 18 μL.

(3) Addition of visible dye

1 μL of the following visible dye was added to the reaction solution prepared in the previous step (2) so that the final concentration in the PCR reaction solution was 0.001% to 0.00001%. 1000 copies / μL of each food poisoning bacterium DNA genome mixture was added to prepare a 20 μL reaction system.

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 the Gut microbiota gene detection kit-probe detection- (Toyobo). As the measuring machine stand, CFX96WELL DEEP manufactured by BioRad was used. TaqMan (R) probes corresponding to Salmonella (Cy5 channel), enterohemorrhagic Escherichia coli (ROX channel), Shigella (HEX channel), and internal standard control (FAM channel) were used to detect each amplified product.

(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 20. The results are shown in FIGS. 1 and 3. Depending on the type of visible dye, a decrease in the ultimate fluorescence intensity was confirmed, but the genomic DNA of 3 bacterial species and 4 types of signals of the internal standard control were detected as positive under all conditions.

Test Example 6. Examination of multiplex detection of genomic DNA derived from microorganisms using PCR reaction solution containing visible dye in the presence of fecal suspension

(1-1) Preparation of genomic DNA derived from microorganisms

As a sample of the genome derived from microorganisms, DNA of each food poisoning-causing bacterium was detected. Three species of Salmonella, Enterohemorrhagic Escherichia coli (Vircell), and Shigella (Zeptomatrix) were mixed and used so as to have a concentration of 500 copies / μL.

(1-2) Preparation of fecal suspension

Negative stool specimens confirmed to be negative for Salmonella, enterohemorrhagic Escherichia coli, and Shigella were suspended in 1 mL of sterile water to prepare a 10% stool suspension. The 10% stool suspension was centrifuged at 15,000 rpm for 5 minutes with a centrifuge to obtain a stool suspension supernatant.

(2) Reaction solution

PCR was performed using the reaction solution having the composition shown below as a basic composition and four types of templates: Salmonella, enterohemorrhagic Escherichia coli, Shigella, and internal standard control.

Reaction solution

(Gut microbiota gene detection kit-probe detection- (Toyobo) attachment)

Primer solution (2 μL of 10x primer solution)

(Gut microbiota gene detection kit-probe detection- (Toyobo) attachment)

Probe solution (2 μL of 10 x probe solution)

(Gut microbiota gene detection kit-probe detection- (Toyobo) attachment)

4.2 ng / μl rTth DNA polymerase (Toyobo)

0.01 μg / μl anti-Tth antibody

Each of the above reagents was mixed to prepare a PCR reaction solution so that the final liquid volume was 17 μL.

(3) Addition of visible dye

1 μL of the following visible dye was added to the reaction solution prepared in the previous step (2) so that the final PCR concentration was 0.001%. 500 copies / μL of each food poisoning bacterium DNA genome mixture was added together with 1 μL and 1 μL of the stool suspension supernatant to prepare a 20 μL reaction system.

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 the Gut microbiota gene detection kit-probe detection- (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 a threshold value of 20. The results are shown in FIGS. 2 and 4. Depending on the type of visible pigment, a decrease in the reached fluorescence intensity was confirmed, but the genomic DNA of 3 strains and 4 types of signals of the internal standard control were positive under all conditions regardless of the presence or absence of fecal suspension. was detected.

Comparative example 1. Examination of detection of microbial-derived genomic DNA multiplex using TaqDNA polymerase in the presence of fecal suspension and visible dye

(1-1) Preparation of genomic DNA derived from microorganisms

As a sample of the genome derived from microorganisms, DNA of each food poisoning-causing bacterium was detected. Three species of Salmonella, Enterohemorrhagic Escherichia coli (Vircell), and Shigella (Zeptomatrix) were mixed and used so as to have a concentration of 500 copies / μL.

(1-2) Preparation of fecal suspension

A 10% stool suspension was prepared with 1 mL of sterile water for negative stool samples confirmed to be negative for Salmonella, enterohemorrhagic Escherichia coli, and Shigella. The 10% stool suspension was centrifuged at 15,000 rpm for 5 minutes with a centrifuge to obtain a stool suspension supernatant.

(2) Reaction solution

PCR was performed using the reaction solution having the composition shown below as a basic composition and four types of templates: Salmonella, enterohemorrhagic Escherichia coli, Shigella, and internal standard control.

Reaction solution

(Gut microbiota gene detection kit-probe detection- (Toyobo) attachment)

Primer solution (2 μL of 10x primer solution)

(Gut microbiota gene detection kit-probe detection- (Toyobo) attachment)

Probe solution (2 μL of 10 x probe solution)

(Gut microbiota gene detection kit-probe detection- (Toyobo) attachment)

4.2 ng / μl rTaq DNA polymerase (Toyobo)

0.01 μg / μl anti-Taq antibody (Toyobo)

Each of the above reagents was mixed to prepare a PCR reaction solution so that the final liquid volume was 17 μL.

(3) Addition of visible dye

1 μL of the following visible dye was added to the reaction solution prepared in the previous step (2) so that the final PCR concentration was 0.001%. 500 copies / μL of each food poisoning bacterium DNA genome mixture was added together with 1 μL and 1 μL of the stool suspension supernatant to prepare a 20 μL reaction system.

Condition 1 Patent Blue

Condition 2 Acid red 18

Condition 3 No visible pigment (sterile water)

(4) PCR reaction

The PCR reaction conditions were the optimum conditions described in the Gut microbiota gene detection kit-probe detection- (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 a threshold value of 20. The results are shown in FIGS. 3 and 5. In the presence of visible dye, the reached fluorescence intensity is reduced, and when the fecal suspension is added, all signals including the internal standard control are negative in the presence of Acid red18, and 3 strains are present in the presence of patent blue. All three types of genomic DNA signals were negative.

Test Example 7. Multiplex detection study of norovirus RNA using TthDNA polymerase in the presence of visible dye (2-enzyme system 1-step RT-PCR reaction)

(1) Preparation of norovirus RNA

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

(2) Reaction solution

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

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 was added to the reaction solution prepared in the previous step (2) so that the final PCR concentration was 0.001% to 0.00001%. 1 μL of 1250 copies / μL of norovirus synthetic RNA was added and added to give a 50 μL reaction system.

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. 4 and 6. 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.

Test Example 8. Multiplex detection study of norovirus sample using TthDNA polymerase in the presence of fecal sample and visible dye (2-enzyme system 1-step RT-PCR reaction)

(1-1) Preparation of norovirus sample

As a sample of norovirus, norovirus samples Norovirus GI and GII Positive Control (ZeptoMetrix, intact) were used. Each sample was prepared to be G1 type and G2 type norovirus, 50 copies / μL.

(1-2) Preparation of fecal suspension

Negative stool specimens confirmed to be negative for norovirus were suspended in 1 mL of sterile water to prepare a 10% stool suspension. The 10% stool suspension was centrifuged at 15,000 rpm for 5 minutes with a centrifuge to obtain a stool suspension supernatant.

(1-3) Pretreatment of norovirus sample and fecal suspension supernatant

1 μL of 50 copies / μL of G1 type and G2 type norovirus sample, 1 μL of fecal suspension supernatant and 3 μL of pretreatment liquid (norovirus detection kit G1 / G2-high-speed probe detection Quick Step- (Toyobo) attachment) at room temperature And mixed to obtain a pretreated sample solution. The pretreatment method performed here does not involve the nucleic acid isolation treatment.

(2) Reaction solution

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

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)

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 44 μL.

(3) Addition of visible dye

1 μL of the following visible dye was added to the reaction solution prepared in the previous step (2) so that the final PCR concentration was 0.001%. 5 μL of the pretreated sample solution was added to prepare a 50 μL reaction system.

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. 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 a threshold value of 50. The results are shown in FIGS. 5 and 7. Depending on the type of visible pigment, a decrease in the ultimate fluorescence intensity was confirmed, but even under the conditions for adding the stool suspension supernatant, two types of norovirus type and three types of internal standard control signals were emitted under all conditions. Detected as positive.

Test Example 9. 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) Pretreatment of SARS-CoV-2 coronavirus sample and saliva

Mix 1 μL of 20 copies / μL of SARS-CoV-2 coronavirus sample with 1 μL of saliva and 3 μL of pretreatment solution (SARS-CoV-2 Detection Kit -N set- (Toyobo) attachment) at 95 ° C for 5 minutes. After heat treatment, it was used as a pretreated sample solution. 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 -N set- (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 final volume was 45 μL.

(2-2) Primer / probe solution

The prama and probe 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.

(3) Addition of visible dye

1 μL of the following visible dye was added to the reaction solution prepared in the previous step (2) so that the final PCR concentration was 0.001%. 5 μL of the pretreated sample solution was added to prepare a 50 μL reaction system.

Condition 1 No visible pigment (sterile water)

Condition 2 Acid red 18

Condition 3 Tartrazine

Condition 4 Patent Blue

Condition 5 BPB

(4) PCR reaction

The PCR reaction conditions were the optimum conditions described in SARS-CoV-2 Detection Kit-N set- (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. 6 and 8. Detection of N1 and N2 regions was confirmed under all conditions even in the presence of saliva.

Test Example 10. Multiplex detection study of norovirus sample using TthDNA polymerase in the presence of fecal sample and visible dye (1 enzyme system 1 step RT-PCR reaction)

(1-1) Preparation of norovirus sample

As a sample of norovirus, norovirus samples Norovirus GI and GII Positive Control (ZeptoMetrix, intact) were used. Each sample was prepared to be G1 type and G2 type norovirus, 50 copies / μL.

(1-2) Preparation of fecal suspension

Negative stool specimens confirmed to be negative for norovirus were suspended in 1 mL of sterile water to prepare a 10% stool suspension. The 10% stool suspension was centrifuged at 15,000 rpm for 5 minutes with a centrifuge to obtain a stool suspension supernatant.

(2) Reaction solution

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

Reaction solution

(RTaq DNA Polymerase 10xBuffer (Toyobo) attachment)

Primer solution (5 μL of 10x primer solution)

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

Probe solution (5 μL of 10 x probe solution)

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

0.2 mM sNTPs Mixture (Toyobo)

2 mM Mn (OAc) 2 (Toyobo)

4.2 ng / μl rTth DNA polymerase (Toyobo)

0.01 μg / μl anti-Tth antibody

3 mg / ml BSA

0.5% fish gelatin

Each of the above reagents was mixed with 1 μL of 50 copies / μL of G1 type and G2 type norovirus samples and 1 μL of stool suspension supernatant so that the final liquid volume was 49 μL to prepare an RT-PCR reaction solution.

(3) Addition of visible dye

1 μL of the following visible dye was added to the reaction solution prepared in the previous step (2) so that the final PCR concentration was 0.001% to prepare a reaction system of 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

A real-time PCR reaction was carried out using CFX96WELL DEEP manufactured by BioRad in the following temperature cycle. The fluorescence value was read in an extension step of 52 ° C. and 40 cycles.

90 ° C for 1 minute (heat treatment conditions)

56 ° C for 5 minutes (reverse transcription conditions)

95 ° C 1 second-52 ° C 10 seconds 10 cycles (PCR)

95 ° C 1 second-52 ° C 10 seconds 40 cycles (PCR-fluorescence reading)

(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. 7 and 9. Depending on the type of visible dye, a decrease in the ultimate fluorescence intensity was confirmed, but under all conditions, two types of norovirus and three types of internal standard control signals were detected as positive.

Test Example 11. Multiplex detection study of norovirus-positive specimens using TthDNA polymerase in the presence of visible dye (1 enzyme system, 1 step RT-PCR reaction)

(1) Preparation of fecal suspension

Fecal samples (15 negative and 30 positive) confirmed to be negative and positive for norovirus were suspended in 1 mL of sterile water to prepare a 10% fecal suspension. The 10% stool suspension was centrifuged at 15,000 rpm for 5 minutes with a centrifuge to obtain a stool suspension supernatant.

(2) Reaction solution

Norovirus was detected by 1-enzyme-based 1-step RT-PCR using the reaction solution having the composition shown below as a basic composition and using the reaction solution under two conditions, one containing the visible dye and the other not containing the visible dye.

Reaction solution

(RTaq DNA Polymerase 10xBuffer (Toyobo) attachment)

Primer solution (5 μL of 10x primer solution)

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

Probe solution (5 μL of 10 x probe solution)

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

0.2 mM sNTPs Mixture (Toyobo)

2 mM Mn (OAc) ₂ (Toyobo)

4.2 ng / μl rTth DNA polymerase (Toyobo)

0.01 μg / μl anti-Tth antibody

3 mg / ml BSA

0.5% fish gelatin

0.0001% or 0% Acid red18

Each of the above reagents and 1 μL of the stool suspension supernatant were mixed so that the final liquid volume was 50 μL to prepare an RT-PCR reaction liquid.

(3) PCR reaction

A real-time PCR reaction was carried out using CFX96WELL DEEP manufactured by BioRad in the following temperature cycle. The fluorescence value was read in an extension step of 52 ° C. and 40 cycles.

90 ° C for 1 minute (heat treatment conditions)

56 ° C for 5 minutes (reverse transcription conditions)

95 ° C 1 second-52 ° C 10 seconds 10 cycles (PCR)

95 ° C 1 second-52 ° C 10 seconds 40 cycles (PCR-fluorescence reading)

(4) 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 Table 10. The number of negative and positive judgments was the same regardless of the presence or absence of the visible dye.

Test Example 12. Multiplex detection study of norovirus specimens using various DNA polymerases in the presence of visible dye (2-enzyme system 1-step RT-PCR reaction)

(1-1) Preparation of norovirus sample

As a sample of norovirus, norovirus samples Norovirus GI and GII Positive Control (ZeptoMetrix, intact) were used. Each sample was prepared to be G1 type and G2 type norovirus, 250 copies / μL.

(1-2) Preparation of fecal suspension

Negative stool specimens confirmed to be negative for norovirus were suspended in 1 mL of sterile water to prepare a 10% stool suspension. The 10% stool suspension was centrifuged at 15,000 rpm for 5 minutes with a centrifuge to obtain a stool suspension supernatant.

(1-3) Pretreatment of norovirus sample and fecal suspension supernatant

1 μL of the fecal suspension supernatant and 3 μL of the pretreatment solution (norovirus detection kit G1 / G2-high-speed probe detection Quick Step- (Toyobo) attachment) were mixed at room temperature to prepare a pretreated sample solution. The pretreatment method performed here does not involve the nucleic acid isolation treatment.

(2) Reaction solution

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

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)

4.2 ng / μL Each DNA polymerase

0.01 μg / μL Each anti-DNA polymerase antibody

0.1U / μL MMLV R Tase (Toyobo)

0.001% Acid red18

Each of the above reagents was mixed to prepare an RT-PCR reaction solution so that the final liquid volume was 45 μL. 4 μL of the pretreated sample solution and 1 μL of norovirus RNA prepared at 250 copies / μL were added to this RT-PCR reaction solution to prepare a total of 50 μL.

(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 (M749K)

Enzyme 5: Tth mutant (F751Y)

Enzyme 6: Tth mutant (D549G)

・ Enzyme 7: HawkZ05 DNA polymerase (Roche)

(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. 8 and 11. As a result, Tth DNA polymerase and its mutants and HawkZ05 DNA polymerase were detected as positive in all noroviruses G1 and G2 even when the sample was added. On the other hand, in TaqDNA polymerase, the rise of the internal standard could not be confirmed when the sample was added.

Test Example 13. 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) Pretreatment of SARS-CoV-2 coronavirus sample and saliva

Mix 1 μL of 20 copies / μL of SARS-CoV-2 coronavirus sample with 1 μL of saliva and 3 μL of pretreatment solution (SARS-CoV-2 Detection Kit -N set- (Toyobo) attachment) at 95 ° C for 5 minutes. After heat treatment, it was used as a pretreated sample solution. 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 -N set- (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)

0.001% Acid red18

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 45 μL.

(2-2) Primer / probe solution

The prama and probe 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-N set- (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. 9 and 12. 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 Taq DNA polymerase.

Test Example 14. Multiplex detection study of norovirus specimens using various DNA polymerases in the presence of visible dye (1 enzyme system, 1 step RT-PCR reaction)

(1-1) Preparation of norovirus sample

As a sample of norovirus, norovirus samples Norovirus GI and GII Positive Control (ZeptoMetrix, intact) were used. Each sample was prepared to be G1 type and G2 type norovirus, 250 copies / μL.

(1-2) Preparation of fecal suspension

Negative stool specimens confirmed to be negative for norovirus were suspended in 1 mL of sterile water to prepare a 10% stool suspension. The 10% stool suspension was centrifuged at 15,000 rpm for 5 minutes with a centrifuge to obtain a stool suspension supernatant.

(2) Reaction solution

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

Reaction solution

(RTaq DNA Polymerase 10xBuffer (Toyobo) attachment)

Primer solution (5 μL of 10x primer solution)

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

Probe solution (5 μL of 10 x probe solution)

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

0.2 mM sNTPs Mixture (Toyobo)

2 mM Mn (OAc) ₂ (Toyobo)

4.2 ng / μl Each DNA polymerase

0.01 μg / μl Each anti-DNA polymerase antibody

3 mg / ml BSA

0.5% fish gelatin

0.001% Acid red 18

The RT-PCR reaction solution was prepared by mixing 1 μL of 250 copies / μL of G1 type and G2 type norovirus samples and 1 μL of stool suspension supernatant with each of the above reagents so that the final liquid volume was 50 μL.

(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 (E628K)

Enzyme 5: Tth mutant (M749K)

Enzyme 6: Tth mutant (F751Y)

Enzyme 7: Tth mutant (D549G)

Enzyme 8: HawkZ05 DNA polymerase (Roche)

(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. 10 and 13. As a result, Tth DNA polymerase and its mutants and HawkZ05 DNA polymerase were detected as positive in all noroviruses G1 and G2. On the other hand, in TaqDNA polymerase, the rise of the internal standard could not be confirmed when the sample was added.

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

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 mixing a sample that has not been isolated from nucleic acid and a PCR reaction solution containing a DNA polymerase having visible dye and contaminant resistance.
(2) A step of carrying out a PCR reaction after sealing the reaction vessel; and (3) a step of detecting two or more target nucleic acids with two or more kinds 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 fecal sample, a saliva sample, a sputum sample, a mouthwash, a tear fluid, a pharyngeal swab sample, a nasal swab 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 (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.
The method according to any one of claims 1 to 12, 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 13, wherein the fluorescent compound 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 method according to claim 14, 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 15, wherein the fluorescent compound is a hybridization probe.
The method according to claim 16, wherein the hybridization probe is a TaqMan probe.
The method according to any one of claims 1 to 17, wherein the two or more kinds of fluorescent compounds are a combination of a hybridization probe and a double-stranded DNA-bound fluorescent compound.
The method according to any one of claims 1 to 18, wherein at least one of the two or more target nucleic acids in the sample is a nucleic acid derived from a pathogenic microorganism.
The method according to claim 19, wherein the pathogenic microorganism is a pathogenic bacterium that causes food poisoning.
The method according to claim 20, wherein the pathogen causing food poisoning is at least one selected from the group consisting of Salmonella, enterohemorrhagic Escherichia coli (EHEC), and Shigella.
The method according to claim 19, wherein the pathogenic microorganism is a DNA virus or an RNA virus.
The method according to claim 22, wherein the RNA virus is an RNA virus having an envelope.
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.
The method according to claim 22, wherein the RNA virus is an RNA virus having no 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.
The method according to claim 27 or 28, wherein the RNA virus having no envelope 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 a divalent cation of 1 mM or more.
The presence or absence of two or more target nucleic acids with two or more fluorescent compounds in a sample not subjected to nucleic acid isolation treatment, which comprises a PCR reaction solution containing a visible dye and a DNA polymerase having contamination resistance. A test kit or composition used for testing.
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 contains 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 33-39, 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, 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 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 kit or composition according to any one of claims 33 to 42, 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 13, wherein the fluorescent compound 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 44, which is one or more selected from the group consisting of 480 ResoLight.
The method according to any one of claims 33 to 45, wherein the fluorescent compound is a hybridization probe.
The kit or composition according to claim 46, wherein the hybridization probe is a TaqMan probe.
The kit or composition according to any one of claims 33 to 47, wherein the two or more kinds of fluorescent compounds are a combination of a hybridization probe and a double-stranded DNA-bound fluorescent compound.