WO2023176026A1

WO2023176026A1 - Test method and test kit

Info

Publication number: WO2023176026A1
Application number: PCT/JP2022/037871
Authority: WO
Inventors: 英明丸瀬; 正光四方; 慎一郎小林; 直子高岡; 健二二宮; 知子稲垣
Original assignee: 株式会社島津製作所
Priority date: 2022-03-16
Filing date: 2022-10-11
Publication date: 2023-09-21

Abstract

This method tests for the presence/absence of the SARS-CoV-2 virus and influenza A/B virus, said method comprising: a step for preparing a mixed liquid by mixing a test specimen processing liquid with a test sample; a step for preparing a PCR solution by mixing the mixed liquid with a buffer solution, an internal standard substance, a SARS-CoV-2 primer, a SARS-CoV-2 probe, an influenza A primer, an influenza A probe, an influenza B primer, an influenza B probe, a reverse transcriptase, and a PCR enzyme; and a step for carrying out PCR processing on the PCR solution and detecting light emitted from at least one of the SARS-CoV-2 probe, the influenza A probe, and the influenza B probe. Moreover, a fluorescent dye with which the influenza A probe is modified and a fluorescent dye with which the influenza B probe is modified are the same.

Description

Test method and test kit

The present invention relates to a method and a test kit for testing SARS-CoV-2 virus and influenza virus.

The gene amplification method (PCR method: Polymerase Chain Reaction) is mainly used to test for the presence or absence of infection with the new coronavirus (SARS-CoV-2 (nCoV)), which causes a very high fever. The National Institute of Infectious Diseases has published the Pathogen Detection Manual 2019-nCoV based on the PCR method (see Non-Patent Document 1). The manual states that samples such as nasal cavity swabs, throat swabs, nasal secretions, nasal washes, sputum, saliva, etc. are used, and that PCR is performed on these samples after pretreatment such as RNA extraction. There is. On the other hand, the PCR method has long been used to test for infection with influenza viruses that cause high fever, and similarly, the Influenza Diagnosis Manual (4th edition, December 2018) based on the PCR method was published. (Refer to Non-Patent Document 2).

As the PCR method, real-time PCR using a hydrolysis probe modified (labeled) with a fluorescent dye and a quenching dye is widely used from the viewpoint of rapidity and quantitative performance. In this method, the amount of nucleic acid amplified by PCR is determined by detecting and analyzing in real time the amplification of light intensity emitted from a probe that is degraded during amplification.

By the way, especially in recent years, there has been a need to simultaneously test patients with fever for infection with the new coronavirus and influenza virus using the PCR method. Furthermore, influenza viruses that infect humans can be broadly classified into two types: influenza virus type A and influenza virus type B, and it is necessary to determine all of these two types of viruses.

Furthermore, in tests using the PCR method, the PCR reaction itself may not occur due to the storage conditions of the test kit or the settings of the device. In this case, even if the target virus is present in the sample, the nucleic acid is not amplified, resulting in a problem in which a negative result is erroneously determined (false negative). Therefore, by including another nucleic acid (internal control) in the test kit, even if the virus to be tested is not present in the sample, the internal control can be amplified and whether or not the PCR reaction is occurring normally can be determined. must be checked to prevent false negatives.

Therefore, in order to simultaneously test for the presence of influenza virus infection in addition to the new coronavirus while preventing false negatives, it is necessary to test for at least four types of infection: the new coronavirus, influenza A, influenza B, and internal control. It was necessary to use different oligonucleotide probes to detect four different types of light.

However, many of the general-purpose PCR devices currently used in hospitals and compatible with conventional influenza tests can only detect three types of light, and the method of detecting the four types of light described above cannot be used.

An object of the present invention is to provide a test method and a test kit that can simultaneously test for the presence or absence of infection with the new coronavirus and influenza virus using a general-purpose PCR device while preventing false negative determinations.

A first aspect of the present invention is a method for testing the presence or absence of SARS-CoV-2 virus and influenza A/B virus, comprising: mixing a specimen processing liquid with a specimen sample to prepare a mixed liquid; A buffer solution, an internal standard substance, a SARS-CoV-2 primer, a SARS-CoV-2 probe, an influenza A primer, an influenza A probe, an influenza B primer, an influenza B probe, a reverse transcriptase, and a PCR enzyme are added to the mixed solution. a step of preparing a PCR solution by performing PCR processing on the PCR solution, and detecting light emitted from at least one of a SARS-CoV-2 probe, an influenza A probe, and an influenza B probe. The fluorescent dye modified on the influenza A probe and the fluorescent dye modified on the influenza B probe are the same.

Further, the test kit according to the first aspect of the present invention is a kit for testing the presence or absence of SARS-CoV-2 virus and influenza virus, and includes a sample processing solution, a buffer solution, an internal standard substance, and SARS-CoV-2 virus. A primer, a SARS-CoV-2 probe, an influenza A primer, an influenza A probe, an influenza B primer, an influenza B probe, a reverse transcriptase, and a fluorescent dye that has a PCR enzyme and is modified into an influenza A probe; The fluorescent dye modified on the B probe is the same.

According to the first aspect of the present invention, it is possible to simultaneously test for infection with influenza virus in addition to the new coronavirus while preventing false negative determinations.

Figure 1 shows a fluorescence amplification curve graph (vertical axis is relative fluorescence units, horizontal axis indicates the number of PCR cycles). SC2 indicates luminescence due to the SARS-CoV-2 probe, FluA/B indicates luminescence due to influenza A probe or influenza B probe, and IC indicates luminescence due to the internal standard probe. FIG. 2 shows a fluorescence amplification curve graph when the testing method of the present invention was performed on a specimen containing only the SARS-CoV-2 virus. FIG. 3 shows a fluorescence amplification curve graph when the testing method of the present invention was applied to a specimen containing only influenza A virus. FIG. 4 shows a fluorescence amplification curve graph when the testing method of the present invention was applied to a specimen containing only influenza B virus. FIG. 5 shows a fluorescence amplification curve graph when the testing method of the present invention was performed on a sample containing all of the SARS-CoV-2 virus, influenza A virus, and influenza B virus.

1. First Embodiment The test method according to the first embodiment of the present invention simultaneously tests for the presence or absence of the new coronavirus (SARS-CoV-2) and influenza A/B. This step includes a mixed liquid preparation step, a PCR solution preparation step, and a PCR step. First, a test kit that can be suitably used in the test method of the first embodiment will be described.

1-1. Test Kit The test kit of the first embodiment includes a processing liquid container (an example of a first container) containing a test processing liquid, a reaction liquid container (an example of a second container) containing a reaction liquid, and a primer. It includes a primer container (an example of a third container) that stores a liquid, and an enzyme container (an example of a fourth container) that stores an enzyme solution.

The test processing solution stored in the processing solution container is a proteolytic solution for extracting (eluting) each RNA contained in the envelope of the coronavirus and influenza virus from the specimen sample collected from the subject.

The test processing liquid only needs to contain an RNA extraction reagent, for example, contains sodium hydroxide. Additionally, commercially available products such as ISOGEN from Nippon Gene Co., Ltd. and Cica Genius (registered trademark) from Kanto Kagaku Co., Ltd. can also be used.

The test processing solution further contains a substrate for DNA synthesis. By containing the substrate in the test treatment solution, the substrate and the PCR enzyme can be stored as separate reagents, and the PCR reaction can be suppressed during storage of the test kit, thereby suppressing a decrease in PCR accuracy. The substrate is deoxynucleotide triphosphate, and specifically includes at least one of a dNTP mix and dUTP (deoxyuridine triphosphate). The dNTP mix is a mixture consisting of dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dCTP (deoxycytidine triphosphate) and dTTP (deoxythymidine triphosphate).

Furthermore, the test processing liquid preferably contains at least one of a chelate and a reducing agent. This makes it possible to reliably extract RNA and perform PCR testing with higher accuracy. Examples of the chelating agent include glycol ether diamine tetraacetic acid (EGTA), ethylene diamine tetraacetic acid, etc., and preferably EGTA. Examples of the reducing agent include dithiothreitol (DTT), dithioerythritol, β-mercaptoethanol, 3-mercapto-1,2-propanediol, 1,2-ethanethiol, thioglycolic acid, and ammonium thioglycolate. , cysteine, glutathione, etc., preferably DTT.

Examples of the solvent used in the test processing solution include aqueous solvents such as purified water, physiological saline, and buffer solutions, organic solvents such as glycerol, and mixed solvents thereof.

The reaction solution contained in the reaction solution container is a solution for normally functioning or improving the PCR reaction, such as buffering, PCR activity in the presence of a PCR inhibitor, inhibition of a PCR inhibitor, and PCR enzyme. It performs at least one function such as activity. The reaction solution preferably contains a buffer and a surfactant.

Examples of the buffer include Tris buffer, phosphate buffer, borate buffer, and Good's buffer (eg, HEPES). Further, the buffer solution may further contain an organic solvent such as glycerol for catalytic activity.

Examples of the surfactant include anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants, preferably anionic surfactants and nonionic surfactants, More preferred are nonionic surfactants. Examples of anionic surfactants include sodium dodecyl sulfate, ammonium dodecyl sulfate, sodium cholate, alkylbenzene sulfonates, and alkyl carboxylates. Examples of nonionic surfactants include polyoxyethylene sorbitan monolaurate (Tween (registered trademark) 20), polyoxyethylene sorbitan monooleate (Tween (registered trademark) 80), and polyoxyethylene pt-octylphenol. (Triton (registered trademark) X-100) and the like. These can be used alone or in combination of two or more. The concentration of surfactant is, for example, 0.05-5% (w/v). By including the surfactant, the PCR reaction functions efficiently even in the presence of a PCR inhibitor. That is, PCR can be reliably performed even on a sample containing impurities such as a PCR inhibitor. Therefore, it is possible to easily perform PCR by directly adding reaction solution, primer solution, and enzyme solution to a mixture of a sample and sample processing solution without purifying the nucleic acid (especially RNA). Can be done.

The reaction solution preferably further contains an inhibition inhibitor. Such inhibitors inhibit, for example, negatively charged substances derived from living organisms that adsorb to DNA polymerase (e.g., certain sugars, dyes), positively charged substances that adsorb to DNA (e.g., certain proteins), etc. A substance that has a neutralizing effect on a substance and suppresses the function of the inhibitor. Thereby, PCR can be performed more reliably even in the presence of a PCR inhibitor. Specifically, the inhibition inhibitor includes, for example, sulfurized polysaccharides such as heparin, dextran sulfate, heparan sulfate, chondroitin sulfate, dermatan, sulfuric acid, funolan, sulfated agarose, carrageenan, porphyran, fucoidan, sulfated curdlan, etc. Salts thereof; examples thereof include polyamines such as ethylenediamine, trimethylenediamine, spermine, spermidine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, and pentaethylenehexamine, and salts thereof. Commercially available products include Ampdirect (registered trademark) and Ampdirect Plus (registered trademark) manufactured by Shimadzu Corporation. These can be used alone or in combination of two or more.

The reaction solution further contains an internal standard nucleic acid (one of the internal standard substances). Internal standard nucleic acids are nucleic acids that are amplified independently of virus-derived nucleic acids, and include SARS-CoV-2 primers, SARS-CoV-2 probes, influenza A primers, influenza A probes, influenza B primers, and influenza B probes. It is not limited as long as it has a sequence that does not cause cross-reactivity with any of the following. The internal standard nucleic acid may be either RNA or DNA, but preferably includes DNA that does not require a reverse transcription reaction. From the viewpoint of improving amplification efficiency, the chain length of the internal standard nucleic acid is preferably 300 bp or less, more preferably 100 bp or less. Specific examples include housekeeping genes such as β-actin and glyceraldehyde phosphate dehydrogenase; for example, artificially synthesized DNA. An internal standard nucleic acid such as an artificially synthesized DNA may be used after being inserted into a plasmid vector.

The reaction solution may contain other additives. For example, from the viewpoint of further activating the DNA polymerase enzyme, potassium chloride (KCl) and magnesium chloride (MgCl ₂ ) may be contained. From the viewpoint of suppressing non-specific amplification, ammonium sulfate may be contained. From the viewpoint of suppressing the reduction in quenching of the quenching dye (BHQ, etc.) caused by the reducing agent (DTT, etc.), oxidized glutathione may be contained. Dimethyl sulfoxide may be contained from the viewpoint of effectively dissociating the secondary structure of DNA and lowering the melting temperature of DNA. These can be used alone or in combination of two or more.

The primer solution contained in the primer container contains a SARS-CoV-2 primer, a SARS-CoV-2 probe, an influenza A primer, an influenza A probe, an influenza B primer, and an influenza B probe. In addition, it further contains an internal standard primer (one of the internal standard substances) and an internal standard probe (one of the internal standard substances).

As the SARS-CoV-2 primer, influenza A primer, influenza B primer, and internal standard primer, known or commercially available primers can be used. Each primer is generally a primer pair consisting of a forward primer and a reverse primer.

The SARS-CoV-2 primer is a primer specific to cDNA generated by reverse transcription reaction of SARS-CoV-2-containing RNA. Specific examples include primers published by the CDC (Centers for Disease control and Prevention), primers published by the National Institute of Infectious Diseases, and primers in Examples described below.

Influenza A primer and influenza B primer are primers specific for cDNA generated by reverse transcription reaction of influenza A or influenza B-containing RNA. Specific examples include primers published by the National Institute of Infectious Diseases, primers in Examples described below, and the like. Note that influenza types A and B include subtypes such as the AH3 subtype, type B Victoria strain, and type B Yamagata strain, and primers specific to cDNA derived from these subtypes may be used. Further, each of these primers can be used alone or in combination of two or more.

The internal standard primer is a primer specific to the sequence of the internal standard nucleic acid, is appropriately determined according to the internal standard nucleic acid, and may be a known or commercially available primer.

The SARS-CoV-2 probe, influenza A probe, influenza B probe, and internal standard probe are hydrolysis probes, and each has an oligonucleotide portion, a reporter portion, and a quencher portion. That is, each probe is an oligonucleotide modified with a fluorescent dye (luminescent dye) and a quenching dye.

The oligonucleotide portion is a portion for hybridizing to the nucleic acid to be detected, and is a polynucleotide in which multiple nucleotides are linearly polymerized. The number of nucleotides is, for example, 5 or more, preferably 10 or more, and is, for example, 50 or less, preferably 35 or less.

The base sequence of the oligonucleotide portion is appropriately determined depending on the nucleic acid region to be hybridized, and a known base sequence can be employed. For example, the SARS-CoV-2 probe, influenza A probe, and influenza B probe include base sequences published by the CDC or the National Institute of Infectious Diseases, probes in Examples described below, and the like. The internal standard probe is appropriately determined depending on the type of internal standard nucleic acid, and a known or commercially available set of the internal standard primer and the internal standard primer can be used. As the primer and probe for such an internal standard, for example, the set used in the 2019 novel coronavirus detection reagent kit manufactured by Shimadzu Corporation may be employed.

The reporter part is a fluorescent dye part that allows the amplification of the target nucleic acid to be detected by emitting fluorescence when released from the probe by hydrolysis. The reporter moiety is attached to the 5' end (or 3' end) of the oligonucleotide moiety.

As the fluorescent dye constituting the reporter part, a known fluorescent dye can be used, and for example, fluorescent dyes commercially available from Funakoshi Co., Ltd., Molecular Probe Co., Ltd., etc. can be used. For example, rhodamine dyes such as ROX (6-carboxy-X-rhodamine); cyanine dyes such as Cy3 (indocarbocyanine-3) and Cy5 (indocarbocyanine-5); FAM (6-carboxyfluorescein), HEX (4, Examples include fluorescein dyes such as 7,2',4',5',7'-hexachloro-6-carboxyfluorescein; examples include xanthene dyes.

In the first embodiment, the fluorescent dye modified on the influenza A probe and the fluorescent dye modified on the influenza B probe are the same. Note that "the fluorescent dyes are the same" means that the main skeletons of these compounds are substantially the same, and the wavelength ranges in which they emit light are substantially the same. On the other hand, the fluorescent dyes modified on the SARS-CoV-2 probe, the fluorescent dyes modified on the influenza A probe (or influenza B probe), and the fluorescent dyes modified on the internal standard probe are mutually exclusive. The wavelength range of light emitted by these fluorescent dyes also differs from each other.

The fluorescent dye modified on each probe can be arbitrarily selected from the fluorescent dyes exemplified above, etc., as long as it meets the above conditions. Preferably, the fluorescent dye modified on the SARS-CoV-2 probe is a rhodamine dye, and the fluorescent dye modified on the influenza A probe and influenza B probe is preferably a fluorescein dye (particularly a carboxyfluorescein dye). The fluorescent dye modified into the internal standard probe is a cyanine dye. This makes it easier to detect the amplification of SARS-CoV-2, which is highly important to identify, since the light emitted by the rhodamine dye is relatively easy to detect. Further, as an example of a preferable emission peak wavelength range, the SARS-CoV-2 probe has a wavelength of 550 to 640 nm (yellow/orange region), and the influenza A probe and influenza B probe have a wavelength of 490 to 550 nm (green region). The internal standard probe is 640-770 nm (red region).

The quencher part is a dye part that quenches fluorescence from the reporter part until the reporter part is released by hydrolysis of the probe. The quencher part is attached to the 3' end (or 5' end) of the oligonucleotide part.

The quenching dye constituting the quencher portion only needs to be able to absorb the light emitted from the fluorescent dye, and any known dye can be used, such as those commercially available from Funakoshi Co., Ltd., Molecular Probe Co., Ltd., etc. Fluorescent dyes can be used. Specific examples include TAMRA (Carboxytetramethylrhodamine: registered trademark), Black Hole Quencher (BHQ, registered trademark) 1, BHQ2, BHQ3, MGB-Eclipse (registered trademark), DABCYL, and the like. The probe of the present invention may have an MGB (Minor Groove Binder) or the like in addition to the above-mentioned sites.

Examples of the solvent used in the primer solution include aqueous solvents such as purified water, physiological saline, and buffer solutions; organic solvents such as glycerol; and mixed solvents thereof.

The enzyme solution contained in the enzyme solution container contains reverse transcriptase and PCR enzyme.

Reverse transcriptase is an enzyme that generates single-stranded complementary DNA (cDNA) using virus (SARS-CoV-2, influenza A, influenza B) RNA as a template. Examples of such reverse transcriptases include RNA-dependent DNA polymerases derived from RNA viruses such as avian myeloblastosis virus, Moloney murine leukemia virus, and human immunodeficiency virus, and mutants thereof.

The PCR enzyme is a DNA polymerase, for example, a thermostable DNA polymerase derived from thermophilic bacteria. Specific examples include Taq DNA polymerase, Tth DNA polymerase, KOD DNA polymerase, Pfu DNA polymerase, and mutants thereof.

Examples of the solvent used in the enzyme solution include the same solvents as those exemplified as solvents used in the primer solution.

1-2. Testing Method The testing method of the first embodiment involves performing PCR using the above test kit. Specifically, a mixed solution preparation step, a PCR solution preparation step, and a PCR step are sequentially provided. Each step will be explained in detail below.

(Mixed liquid preparation process)
In this step, a specimen processing liquid is mixed with the specimen sample. In this way, a mixed solution is prepared. This step is a pretreatment for PCR and is carried out to elute RNA contained within each virus.

The specimen sample is a biological sample collected from a subject, and may be, for example, blood, nasal swab, throat swab, nasal discharge, nasal wash, sputum, saliva, urine, feces, etc. The specimen sample may be diluted by appropriately adding purified water, physiological saline, or the like.

After mixing, incubation is preferably performed from the viewpoint of ensuring RNA elution. The temperature is, for example, 40°C or higher, preferably 60°C or higher, and, for example, 100°C or lower, preferably 95°C or lower. The heating time is, for example, 30 seconds or more, preferably 1 minute or more, and is, for example, 20 minutes or less, preferably 10 minutes or less.

As a result, the membranes (envelope, cell membrane, etc.) possessed by SARS-CoV-2, influenza A, and influenza B are dissolved in the mixture, and as a result, SARS-CoV-2 RNA, influenza A RNA, and Influenza B RNA is exposed.

Incidentally, a supernatant liquid may be collected from the mixed liquid using a centrifuge, and this supernatant liquid may be used in the next step. Furthermore, if necessary, cooling on ice or the like may be performed appropriately after incubation.

(PCR solution preparation process)
In this step, a reaction solution, a primer solution, and an enzyme solution are mixed into the mixed solution. In this way, a PCR solution is prepared.

Regarding the mixing order, the reaction solution, primer solution, and enzyme solution may be added to the mixed solution sequentially or at the same time.Also, after preparing a masterbatch in which the reaction solution, primer solution, and enzyme solution are mixed, the reaction solution, primer solution, and enzyme solution may be added to the mixed solution. A masterbatch may also be added.

In addition to mixed solution components such as eluted RNA, the prepared PCR solution contains buffer, surfactant, internal standard substances (internal standard nucleic acid, internal standard primer, internal standard probe), and SARS-CoV-2 primer. , SARS-CoV-2 probe, influenza A primer, influenza A probe, influenza B primer, influenza B probe, reverse transcriptase and PCR enzyme.

(PCR process)
In this step, PCR processing is performed on the PCR solution, and light amplified by the PCR processing is detected. This makes it possible to determine the presence or absence of the SARS-CoV-2 virus and influenza A/B, and also to confirm false negatives.

Specifically, real-time PCR method is performed. That is, the intensity of light amplified by PCR is measured in real time on a monitor. Specifically, each time a virus-derived cDNA and internal standard nucleic acid are amplified by performing PCR, probes hybridized to the nucleic acids (SARS-CoV-2 probe, influenza A probe, influenza B probe, internal The standard probe) is decomposed, and the amount of fluorescent dye released from the quenching dye (as a result, the luminescence intensity) increases. This luminescence intensity is distinguished for each emission wavelength of the probe and continues to be detected in real time, and is output and observed on a monitor as a graph showing the relationship between the number of PCRs and the luminescence intensity.

A known or commercially available PCR device can be used for the real-time PCR method. In the first embodiment, an apparatus is used that can detect at least three types of fluorescent dyes (that is, the number of detectable emission wavelengths). For example, any device that can distinguish and simultaneously detect three types of light emitted from ROX, FAM, and Cy5 may be used.

The reaction conditions of the real-time PCR method, such as temperature, time, and number of PCR cycles, can be carried out according to conventional methods. For example, it can be carried out according to the settings built into a commercially available real-time PCR analyzer.

If the sample contains SARS-CoV-2, an amplification curve in which light in the wavelength range emitted by the fluorescent dye of the SARS-CoV-2 probe is amplified is observed on the monitor, so SARS-CoV-2 The presence or absence of CoV-2 infection can be determined. If the sample contains at least one of influenza A and influenza B, an amplification curve is observed in which light in the wavelength range emitted by the fluorescent dye of the influenza A probe (or influenza B probe) is amplified. Therefore, the presence or absence of influenza infection can be determined. In particular, both type A and type B influenza infections can be determined without exception, without distinguishing between type A and type B influenza.

On the other hand, an amplification curve in which light in the wavelength range emitted by the fluorescent dye of the internal standard probe is amplified is not shown unless the PCR reaction is not functioning normally because the test kit contains the internal standard nucleic acid. , observed. Therefore, it is possible to avoid a false negative determination of the virus due to a defect in the PCR reaction.

According to the testing method of the first embodiment of the present invention, it is possible to test for SARS-CoV-2 and influenza infection using a conventional general-purpose PCR device while preventing false negatives.

2. Other Embodiments In the test kit and test method of the first embodiment, a combination consisting of an internal standard nucleic acid, an internal standard primer, and an internal standard probe was described as an example of the internal standard substance included in the test kit. The test kit does not contain an internal standard nucleic acid, and the internal standard substance contained in the test kit may be a combination of an internal standard primer and an internal standard probe. In this case, the internal standard nucleic acid may be a housekeeping gene contained in the specimen sample, and the internal standard primer and internal standard probe may be selected from primers and probes specific to the housekeeping gene.

3. Aspects Those skilled in the art will appreciate that the exemplary embodiments described above are specific examples of the following aspects.

(Paragraph 1) The test method according to one aspect is a method for testing the presence or absence of SARS-CoV-2 virus and influenza A/B virus, in which a sample processing liquid is mixed with a sample sample to prepare a mixed liquid. and adding a buffer solution, an internal standard substance, a SARS-CoV-2 primer, a SARS-CoV-2 probe, an influenza A primer, an influenza A probe, an influenza B primer, an influenza B probe, a reverse transcriptase to the mixed solution, and a step of preparing a PCR solution by mixing a PCR enzyme, and performing PCR processing on the PCR solution, and emitting light from at least one of a SARS-CoV-2 probe, an influenza A probe, and an influenza B probe. The fluorescent dye modified on the influenza A probe and the fluorescent dye modified on the influenza B probe may be the same.

(Section 2) In the testing method described in Section 1, the SARS-CoV-2 probe may be an oligonucleotide modified with a rhodamine dye.

(Section 3) In the testing method described in Section 1 or 2, the influenza A probe and influenza B probe may be oligonucleotides modified with a fluorescein dye.

(Section 4) In the testing method according to any one of Items 1 to 3, the sample processing liquid may contain deoxynucleotide triphosphate.

(Section 5) In the testing method according to any one of Items 1 to 4, the sample processing liquid may contain deoxynucleotide triphosphate.

(Section 6) In the testing method described in any one of Items 1 to 5, the PCR solution may further contain a surfactant.

(Section 7) The test kit according to one embodiment is a kit for testing the presence or absence of SARS-CoV-2 virus and influenza virus, and includes a sample processing solution, a buffer, an internal standard material, a SARS-CoV-2 primer, SARS-CoV-2 probe, influenza A primer, influenza A probe, influenza B primer, influenza B probe, reverse transcriptase, and a fluorescent dye that has a PCR enzyme and is modified into an influenza A probe, and an influenza B probe. The fluorescent dyes modified in may be the same.

(Section 8) In the test kit according to Section 7, the test processing liquid is contained in the first container,
The buffer solution is contained in a second container, and the SARS-CoV-2 primer, SARS-CoV-2 probe, influenza A primer, influenza A probe, influenza B primer, and influenza B probe are contained in a third container and are inverted. The transcription enzyme and the PCR enzyme may be housed in the fourth container.

Next, the present invention will be explained in detail with reference to Examples, but the scope of the present invention is not limited by these.

<Example 1>
Mix the components listed in Table 1 below in appropriate proportions to prepare the test treatment solution, reaction solution, primer solution, and enzyme solution, and then put these into separate vials (first to fourth containers). Contained.

The sequences of various primers and probes (manufacturer: Hokkaido System Science Co., Ltd.) are shown in Table 2. As an internal control, a plasmid (manufacturer: Eurofin Phenomics) in which the following tag (internal standard nucleic acid) was introduced into a plasmid vector (pTAKN-2) was used.

Fluorescent dyes and quenching dyes modified at the 5' or 3' ends of the SARS-CoV-2 probe, influenza A probe, influenza B probe, and internal standard probe are shown below. Note that the peak wavelength of ROX was 599 nm, the peak wavelength of FAM was 520 nm, and the peak wavelength of Cy5 was 667 nm. The combinations of fluorescent dyes and quenching dyes modified on each probe are shown in Table 3 below.

A nasopharyngeal swab from a test subject who was not infected with both the new coronavirus and the influenza virus was mixed with the test treatment solution in a PCR tube and heated at 90°C for 5 minutes.

Next, the reaction solution, primer solution, and enzyme solution were mixed using a vortex mixer to prepare a master mix. This master mix was added to a PCR tube containing the above-mentioned heated treatment solution to prepare a PCR solution. A PCR tube containing a PCR solution was set in a real-time PCR device, and a PCR method was performed.

The conditions for the PRC method were heating at 42°C for 10 minutes and 1 minute at 95°C, followed by 45 PCR cycles of 95°C for 5 seconds and 55°C for 30 seconds. The results are shown in FIG.

<Example 2>
(Only available for SARS-CoV-2)
Same as above except that 20 copies of SARS-CoV-2 were added to the subject's nasopharyngeal swab. The results are shown in FIG.

<Example 3>
(Influenza A virus only)
The procedure was the same as above, except that 40 copies of influenza A virus were added to the nasopharyngeal swab of the subject. The results are shown in FIG.

<Example 4>
(Influenza B virus only)
The same procedure as above was performed except that 20 copies of influenza B virus were added to the nasopharyngeal swab of the subject. The results are shown in FIG.

<Example 5>
(Contains 3 types of viruses)
The procedure was the same as above, except that 20 copies of SARS-CoV-2 virus, 40 copies of influenza A virus, and 20 copies of influenza B virus were added to the subjects' nasopharyngeal swabs. The results are shown in FIG.

<Consideration>
As is clear from FIG. 1, in the test method of the example, false negatives can be prevented by observing the amplification curve using the internal standard nucleic acid. Furthermore, from FIGS. 2 to 5, it is possible to distinguish between SARS-CoV-2 virus infection and influenza (influenza A virus or influenza B virus, or both) infection.

Claims

A method of testing for the presence of SARS-CoV-2 virus and influenza virus, comprising:
A step of preparing a mixed solution by mixing a sample processing solution with a sample sample;
A buffer solution, an internal standard substance, a SARS-CoV-2 primer, a SARS-CoV-2 probe, an influenza A primer, an influenza A probe, an influenza B primer, an influenza B probe, a reverse transcriptase, and a PCR enzyme are added to the mixed solution. a step of preparing a PCR solution by mixing;
A step of performing PCR processing on the PCR solution and detecting light emitted from at least one of a SARS-CoV-2 probe, an influenza A probe, and an influenza B probe,
A testing method in which the fluorescent dye modified on the influenza A probe and the fluorescent dye modified on the influenza B probe are the same.
The testing method according to claim 1, wherein the SARS-CoV-2 probe is an oligonucleotide modified with a rhodamine dye.
The testing method according to claim 1, wherein the influenza A probe and the influenza B probe are oligonucleotides modified with a fluorescein dye.
The test method according to claim 1, wherein the specimen processing liquid contains deoxynucleotide triphosphate.
The test method according to claim 1, wherein the specimen processing liquid further contains at least one of glycol ether diamine tetraacetic acid and dithiothreitol.
The testing method according to claim 1, wherein the PCR solution further contains a surfactant.
A kit for testing the presence of SARS-CoV-2 virus and influenza virus, comprising:
Sample processing solution, buffer solution, internal standard substance, SARS-CoV-2 primer, SARS-CoV-2 probe, influenza A primer, influenza A probe, influenza B primer, influenza B probe, reverse transcriptase, and PCR enzyme. have,
A test kit in which the fluorescent dye modified on the influenza A probe and the fluorescent dye modified on the influenza B probe are the same.
The test processing liquid is contained in the first container,
a buffer solution is contained in a second container;
SARS-CoV-2 primer, SARS-CoV-2 probe, influenza A primer, influenza A probe, influenza B primer and influenza B probe are housed in a third container;
The test kit according to claim 7, wherein the reverse transcriptase and the PCR enzyme are contained in the fourth container.