JP6679852B2 - Method for detecting nucleic acid and reagent kit using the method - Google Patents

Description

  The present invention relates to a method for detecting a target nucleic acid contained in a sample and a reagent kit using the method. More specifically, the present invention relates to a method and reagent kit for detecting a target nucleic acid derived from a virus contained in a sample.

  Testing for viruses in the environment or in samples is routinely performed in the fields of public health and clinical testing. In the field of clinical testing, as a method for testing a virus contained in a sample, an immunochromatographic method using an antibody capable of specifically binding to a protein derived from the virus has been conventionally performed. Although the immunochromatographic test can be performed easily and quickly, the detection sensitivity is low and the false negative has been a problem.

  On the other hand, as a method for detecting a virus contained in a sample with high sensitivity, a nucleic acid derived from the virus is subjected to a PCR method, a NASBA (Nucleic Acid Sequence Based Amplification) method, a TMA (Transcription-Mediated Amplification) method, and a TRC (Transcription-Transversion) method. There is known a method of specifically amplifying the amplified nucleic acid by a method such as the transcription conversion reaction) and detecting the amplified nucleic acid. However, this method was inferior in rapidity to the method using the immunochromatography method described above. Furthermore, this method requires a special device (centrifuge, etc.) for extracting nucleic acids derived from viruses and a dedicated device (a thermal cycler, etc.) for amplifying the extracted nucleic acids. Local implementation was difficult.

  In order to solve the above problems, there is also known a method of amplifying and detecting the nucleic acid without performing an operation of extracting and purifying the nucleic acid, such as the method described in Colony direct PCR (Non-Patent Document 1) and Non-Patent Document 2. Has been. However, these methods were insufficient in terms of detection sensitivity.

Frostingham, R .; et al, Biotechniques, 11, 40-44 (1991). Takeshi Matsumura, The University of Tokyo Dissertation, Report No. 122590 (2007)

  An object of the present invention is a method for detecting a virus derived from a virus contained in a sample by amplifying the nucleic acid, which is simple, rapid, highly sensitive and specific without using a special device. It is intended to provide a method capable of detecting a virus and a reagent kit using the method.

  The present inventors have arrived at the present invention as a result of intensive studies to solve the above problems.

That is, the first aspect of the present invention is a method for detecting a virus having an envelope, which includes the following steps (1) to (3).
(1) a step of contacting a sample containing a virus having an envelope with a solution containing at least a water-soluble organic solvent to extract nucleic acid derived from the virus (2) a specific base sequence in the nucleic acid derived from the virus A solution containing a primer having a sequence homologous to a part, a primer having a sequence complementary to a part of the specific base sequence, and a nucleic acid amplification enzyme is reacted with the nucleic acid extracted in the step (1). A step of amplifying a nucleic acid containing the specific base sequence or a sequence complementary to the specific base sequence (3) An oligo to which a labeling substance capable of hybridizing with the nucleic acid amplified in the step (2) under stringent conditions is bound. The amplified nucleic acid is a nucleotide and an oligonucleotide bound to a solid phase capable of hybridizing with the amplified nucleic acid under stringent conditions. The step of detecting the virus-derived nucleic acid by capturing, and the second aspect of the present invention is a solution containing a water-soluble organic solvent to be brought into contact with a sample containing a virus having an envelope in the step (1), The detection method according to the first aspect, which is a solution containing dimethyl sulfoxide.

  A third aspect of the present invention is that the solution containing the water-soluble organic solvent that is brought into contact with the sample containing the virus having an envelope in the step (1) is a solution further containing one or more kinds of surfactants, It is the detection method according to the second aspect.

  In a fourth aspect of the present invention, the solid phase in the step (3) is a membrane, and the nucleic acid amplified in the step (2) is captured by chromatography using a developing solution. The detection method according to any one of the third aspect.

  A fifth aspect of the present invention is the detection method according to the fourth aspect, wherein the developing solution contains at least a nonionic surfactant.

  A sixth aspect of the present invention is the detection method according to any one of the first to fifth aspects, wherein the virus having an envelope is an influenza virus.

Furthermore, a seventh aspect of the present invention is a virus detection reagent kit having an envelope, which contains the reagents shown in (A) to (E) below.
(A) A reagent for extracting an enveloped virus-derived nucleic acid containing at least dimethyl sulfoxide and one or more kinds of surfactants (B) a sequence homologous to a part of a specific base sequence in the virus-derived nucleic acid A reagent (C) for amplifying a nucleic acid containing the specific base sequence or a complementary sequence of the specific base sequence, which contains at least a primer having, a primer having a sequence complementary to a part of the specific base sequence, and a nucleic acid amplification enzyme Membrane bound to the amplified nucleic acid and an oligonucleotide hybridizable under stringent conditions (D) Oligonucleotide bound to the amplified nucleic acid and a labeling substance capable of hybridizing under stringent conditions (E) Nonionic interface A developing solution for chromatography containing at least an activator The eighth aspect of the present invention The embodiment (4) is the reagent kit according to the seventh embodiment, wherein (E) is a mixture of (A) and (B).

  Hereinafter, the present invention will be described in detail.

  In the present invention, the virus having an envelope refers to a virus in which a viral capsid is covered with an envelope mainly composed of lipids, and examples thereof include herpes simplex virus, influenza virus, RS virus, AIDS virus (HIV). Can be given. Among them, influenza virus is preferable as the detection target virus in the detection method of the present invention.

  In the present invention, the sample containing a virus includes a sample obtained by simply dissolving the virus in water, physiological saline or a buffer solution, a biological sample itself containing the virus, a filter paper containing the biological sample, a swab and the like. Examples of the biological sample include blood, feces, urine, sputum, lymph, plasma, ejaculate, lung aspirate, cerebrospinal fluid, pharyngeal swab, nasal swab, mouthwash, saliva, and tears. Further, a filter paper or a swab, etc., which has been wiped with the surface of soil containing virus or the environmental water containing virus is also included in the sample containing virus in the present invention.

  In the present invention, when the nucleic acid derived from the virus having an envelope is extracted, it is contacted with a solution containing at least a water-soluble organic solvent. The water-soluble organic solvent is an organic solvent having solubility in water under any conditions, and examples thereof include ethanol, methanol, acetone, phenol, and dimethyl sulfoxide (DMSO). Above all, it is preferable to use DMSO as the water-soluble organic solvent because the obtained solution containing the nucleic acid derived from the virus can be used as it is in the nucleic acid amplification step described below. The content of the water-soluble organic solvent may be appropriately set in consideration of the solubility of the solvent used.

  The solution containing at least a water-soluble organic solvent used in the extraction of nucleic acids derived from viruses having an envelope, enzymes, salts such as magnesium salts and potassium salts, sugars such as trehalose, sugar alcohols such as glycerol, and nucleic acids. You may further contain a component, surfactant, etc. Among them, the surfactant is preferable because it has an effect of promoting extraction of viral nucleic acid. Examples of the surfactants to be further contained in the extraction reagent of the present invention include anionic surfactants such as sodium dodecyl sulfate (SDS), sodium cholate, sodium deoxycholate, sodium tauroursodeoxycholate, and CHAPS. Amphoteric surfactants, n-Dodecyl-β-D-maltoside, n-Octyl-β-D-glucoside, Span 20 (trade name), MEGA-8 (trade name), Tween 20 (trade name), Tween. Nonionic surfactants such as 40 (trade name), Tween 60 (trade name), and Tween 80 (trade name) are listed. Among them, SDS, sodium cholate, sodium deoxycholate and sodium tauroursodeoxycholate have the effect of promoting the extraction of virus-derived nucleic acids, and the nucleic acid amplification step described below by bringing in a solution from which virus-derived nucleic acids have been extracted. It is preferable because it has little effect on The surfactant added to the solution containing at least the water-soluble organic solvent may be one kind or a combination of plural kinds. As an example of the combination of the surfactants to be added, a combination of sodium deoxycholate which is an anionic surfactant and Tween 80 (trade name) which is a nonionic surfactant can be exemplified.

  The contact time between the sample containing the virus having an envelope and the solution containing at least the water-soluble organic solvent is determined in consideration of the properties of the sample containing the virus, the virus concentration in the sample, the components contained in the extraction reagent, etc. In most cases, the nucleic acid derived from the virus can be sufficiently extracted within 4 minutes after the contact, and the nucleic acid can be directly subjected to the nucleic acid amplification step described below after the contact.

  In the present invention, after the nucleic acid derived from the virus contained in the sample is extracted by the method described above, the nucleic acid containing the specific base sequence in the nucleic acid or the complementary sequence of the specific base sequence is amplified. In the present invention, the specific base sequence is a region of homology with a primer (hereinafter, also referred to as a first primer) having a sequence homologous to a part of the virus-derived nucleic acid in the virus-derived nucleic acid to be detected. It refers to the base sequence from the 5'end to the 3'end of the complementary region with the primer having a sequence complementary to a part of the virus-derived nucleic acid (hereinafter also referred to as the second primer). That is, in the present invention, a nucleic acid containing the specific base sequence or the complementary sequence of the specific base sequence is amplified.

  To amplify a nucleic acid containing a specific base sequence or a complementary sequence of a specific base sequence in the present invention, a reagent containing the above-mentioned two kinds of primers and a nucleic acid amplification enzyme, and a sample containing the nucleic acid extracted by the above-mentioned method are used. The reaction may be carried out under constant temperature conditions. As the nucleic acid amplification enzyme, an enzyme suitable for the nucleic acid amplification method to be used (for example, PCR method, asymmetric PCR method, RT-PCR method, NASBA method, TMA method, TRC method) may be appropriately used. When amplifying a nucleic acid containing a specific base sequence or a complementary sequence of a specific base sequence using a method for amplifying RNA at a constant temperature such as NASBA method, TMA method, TRC method, either of the above-mentioned two types of primers is used. It is preferable to further add an RNA polymerase promoter to one 5 ′ end side. As a promoter to be added to the primer, a promoter corresponding to an RNA polymerase used for amplification of the nucleic acid (for example, T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase widely used in the field of molecular biology) may be used. A transcription initiation region known to affect transcription efficiency may be further added to the promoter. As a specific example of the promoter (T7 promoter) added to the 5′-terminal side of the primer when T7 RNA polymerase is used as the RNA polymerase used for amplification of the nucleic acid, an oligonucleotide having the sequence set forth in SEQ ID NO: 8 can be mentioned. To be

  In the present invention, after amplifying a nucleic acid containing a specific base sequence or a complementary sequence of the specific base sequence in a nucleic acid derived from a virus contained in a sample by the method described above, the nucleic acid is subjected to stringent conditions with the nucleic acid. The nucleic acid derived from the virus is detected by capturing with two kinds of oligonucleotides that can hybridize with each other (one is bound with a labeling substance and the other is bound to a solid phase) (hereinafter, the labeling substance is bound. The oligonucleotide to be allowed is also referred to as a first probe, and the oligonucleotide bound to the solid phase is also referred to as a second probe). Examples of stringent conditions in the present invention include 50% (v / v) formamide, 0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate at 42 ° C. Examples include conditions in which a buffer (pH 6.5), 150 mM sodium chloride, and 75 mM sodium citrate are present, and the nucleic acid capture conditions described in Examples of the present specification. That is, under the above-mentioned stringent conditions, the base sequences of the two types of probes are not necessarily completely complementary sequences with the nucleic acid, as long as they can hybridize with the base sequence sufficiently specifically and with high efficiency. It does not have to be present, and the base sequence may have substitutions, deletions, additions or modifications.

  The labeling substance to be bound to the first probe in the present invention is a substance that can be detected by the amplified nucleic acid being captured by the first probe and the second probe, and collecting a certain amount of the first probe on the solid phase. It suffices if it is present, and examples thereof include colored polymer particles, colloidal gold particles, dyes, and fluorescent substances. In particular, it is preferable to use a substance that absorbs or reflects visible light as the labeling substance because a certain amount of the labeling substance can be collected and can be visually detected. The binding between the first probe and the labeling substance can be appropriately selected depending on the mode of the probe, and examples thereof include covalent bond, electrostatic bond, antigen-antibody reaction, ligand-receptor bond, biotin-avidin bond, and internucleotide hybrid. Soybean, or a combination thereof may be mentioned.

  In the present invention, the solid phase to be bound to the second probe is a substance that is in a separable state with respect to a solution containing the nucleic acid amplified in the nucleic acid amplification step described above, and if it can be directly or indirectly supported on the probe. The substance is not particularly limited as long as it is a substance, but when a porous membrane such as nitrocellulose or nylon is used as a solid phase, the amplified nucleic acid can be captured by the first probe and the second probe by chromatography using a developing solution. Therefore, it is preferable. In order to strengthen the bond between the second probe and the solid phase, the solid phase may be appropriately processed or a functional group or a binding factor may be introduced. The binding between the second probe and the solid phase can be appropriately selected depending on the aspect of the probe, and examples thereof include covalent binding, electrostatic binding, antigen-antibody reaction, ligand-receptor binding, biotin-avidin binding, and internucleotide hybridization. Soybean, or a combination thereof may be mentioned. The shape of the solid phase is not particularly limited as long as it is in a shape that allows sufficient contact with the reaction solution, and examples thereof include a thin film shape, a cube, and a sphere. Further, the shape may also serve as a part of the inner wall of the reaction container.

  Chromatography in the present invention is a method of moving and developing a solution in a certain direction by capillary phenomenon, and thin film chromatography, paper chromatography, capillary chromatography and the like can be exemplified, and a water-absorbent substrate for development, a developing solution, and development Examples thereof include a kit type having both layers and a dip stick type. When the amplified nucleic acid is captured by the first probe and the second probe by chromatography using a developing solution, the developing solution preferably contains a nonionic surfactant. This is because the separation by chromatography is promoted by adding a nonionic surfactant to the developing solution. The nonionic surfactant can be appropriately selected from nonionic surfactants commonly used by those skilled in the art, and examples thereof include Tween 20, Tween 60, Tween 80, and Triton X-100 (trade name). . Among them, Tween 20 is preferable as the nonionic surfactant added to the developing solution.

The detection reagent kit of the present invention comprises a step of extracting the above-mentioned virus-derived nucleic acid, a step of amplifying a nucleic acid containing a specific nucleic acid sequence in the nucleic acid or a complementary sequence of the specific base sequence, and detecting the amplified nucleic acid. It suffices if it contains a component capable of performing the step, and as one aspect thereof,
A reagent for extracting nucleic acid from a virus having an envelope, containing at least dimethyl sulfoxide and one or more kinds of surfactants, and
A primer having a sequence homologous to a part of the specific base sequence in the virus-derived nucleic acid (first primer), and a primer having a sequence complementary to a part of the specific base sequence (second primer) A reagent for amplifying a nucleic acid containing the specific base sequence or a sequence complementary to the specific base sequence, containing at least a nucleic acid amplification enzyme;
A membrane to which an oligonucleotide (second probe) capable of hybridizing with the amplified nucleic acid under stringent conditions is bound,
An oligonucleotide (first probe) bound with a labeling substance capable of hybridizing with the amplified nucleic acid under stringent conditions,
A developing solution for chromatography containing at least a nonionic surfactant,
A detection reagent kit containing In the above aspect, the first probe may be added to the developing solution or may be applied to the membrane. Further, if the mixed solution of the extraction reagent and the nucleic acid amplification reagent contains a nonionic surfactant, the mixed solution may be used as it is as a developing solution, and the mixed solution may contain the nonionic surfactant. If not, a developing solution may be prepared by adding a nonionic surfactant to the mixed solution before performing chromatography after the nucleic acid amplification reaction, and after the mixed solution is spotted on one end of the membrane, Chromatography may be performed using a new developing solution containing a surfactant.

  When using the reagent kit of the present invention to separately detect different viruses or different types of the same virus (for example, A / B in the case of influenza virus), at least the sequence of the second probe should be used for each virus or each virus. It may be designed to have a sequence that can be captured in a type-specific manner (that is, capable of hybridizing under stringent conditions), and the binding positions of the second probe to the membrane may be different from each other. In addition, it is preferable that the sequence of the first probe is a sequence that can be specifically captured by each virus or each type (that is, capable of hybridizing under stringent conditions) from the viewpoint of improving the specificity, and bind to the first probe. It is preferable that the labeling substance is a substance different for each virus or type (for example, different dye), because it is easy to identify the presence or absence of each virus or type.

  The method for detecting a virus having an envelope of the present invention comprises: (1) a step of contacting a sample containing the virus with a solution containing at least a water-soluble organic solvent to extract the nucleic acid derived from the virus; A primer having a sequence homologous to a part of a specific base sequence in a nucleic acid derived from a virus, a solution containing a primer having a sequence complementary to a part of the specific base sequence, and a nucleic acid amplification enzyme; A step of reacting with the nucleic acid extracted in the step to amplify a nucleic acid containing the specific base sequence or a complementary sequence of the specific base sequence, and (3) under stringent conditions with the nucleic acid amplified in the step (2). An oligonucleotide bound to a hybridizable labeling substance and an oligo bound to a solid phase capable of hybridizing with the amplified nucleic acid under stringent conditions A method comprising a step of visually detecting the nucleic acid derived from the virus by capturing the amplified nucleic acid with cleotide, and by the method of the present invention and a detection reagent kit utilizing the method, high specificity and detection sensitivity are obtained. The virus having the enveloped virus contained in the sample can be detected without using a special device.

It is a figure (chromato strip) which shows the result of having detected the subtype A H3N2 influenza virus by the detection method of this invention. It is a figure (chromato strip) which shows the result of having detected the influenza B virus by the detection method of this invention.

  Hereinafter, the present invention will be described in detail based on examples when an influenza virus is used as a virus having an envelope, but the present invention is not limited to these examples.

Example 1 Preparation of Oligonucleotide (First Probe) Bound with Labeling Substance As a labeling substance, latex particles Estapor (manufactured by Merck) having a diameter of 500 nm were used, and the labeling substance was treated with influenza virus RNA or It was bound to an oligonucleotide (first probe) capable of hybridizing with an internal standard nucleic acid under stringent conditions.
(1) To 1.4 × 10 ¹⁰ latex particles, 150 ng of each of the oligonucleotides having the sequences shown in SEQ ID NOs: 1 to 4 in which the 3 ′ end side was modified with an amino group was added, and the mixture was allowed to stand at room temperature for 30 minutes. did. Note that SEQ ID NO: 1 is the sequence of an oligonucleotide capable of capturing the A type H1N1 subtype influenza virus RNA, SEQ ID NO: 2 is the sequence of an oligonucleotide capable of capturing the type A H3N2 subtype influenza virus RNA, and SEQ ID NO: 3 Is an oligonucleotide sequence capable of capturing influenza B virus RNA, and SEQ ID NO: 4 is an oligonucleotide sequence capable of capturing an internal standard nucleic acid.
(2) Further, water-soluble carbodiimide was added so that the final concentration was 10 mg / mL, and the mixture was reacted at room temperature for 2 hours to bond latex particles to the oligonucleotide.
(3) The precipitate obtained by centrifugation (the first probe bound with the labeling substance) was washed with a solution containing Tween 20 and SDS, and buffered (0.5 mM EDTA, 1M NaCl, and 0.05% Tween 20). 10 mM Tris-HCl (pH 7.4)) containing

Example 2 Preparation of Solid Phase (Second Probe) Bound with Oligonucleotide As a solid phase, a nitrocellulose membrane HF120 (Merck) bound to a mount (Merck) was used, and the following method was used. An oligonucleotide (second probe) capable of hybridizing with influenza virus RNA or an internal standard nucleic acid under stringent conditions was bound to the membrane.
(1) 50 μM of the oligonucleotide having the sequences shown in SEQ ID NOs: 5 to 7 was applied to the membrane using a coating machine. In addition, SEQ ID NO: 5 is a sequence of an oligonucleotide capable of capturing type A (H1N1 subtype and H3N2 subtype) influenza virus RNA, and SEQ ID NO: 6 is a sequence of an oligonucleotide capable of capturing type B influenza virus RNA, SEQ ID NO: 7 is a sequence of an oligonucleotide capable of capturing an internal standard nucleic acid.
(2) After irradiating with UV for 2 minutes, the oligonucleotide was bound to the membrane by drying at 37 ° C. for 2 hours.

Example 3 Detection of influenza virus by the detection method of the present invention An attempt was made to detect influenza virus contained in a sample using the detection method of the present invention.
(1) Type A H3N2 subtype influenza virus (A / Texas / 50/2012, manufactured by ZeptoMetrix) was used in water for injection at 5 × 10 ⁰ TCID ₅₀ / mL, 5 × 10 ⁻¹ TCID ₅₀ / mL and 5 ×. The influenza A virus sample of each concentration was prepared by diluting to 10 ⁻² TCID ₅₀ / mL, and the influenza B virus (B / Massachusetts / 2/2012, manufactured by ZeptoMetrix) was used with water for injection. Samples of influenza B virus at each concentration were prepared by diluting to 5 × 10 ¹ TCID ₅₀ / mL and 5 × 10 ⁰ TCID ₅₀ / mL.
(2) A pharyngeal swab solution was prepared by rubbing the pharyngeal part with a FLOQ swab (manufactured by Shin Corporation) and suspending it in 100 μL of water for injection.
(3) To 11 μL of each virus sample prepared in (1), 0.75 μL of the pharyngeal swab solution prepared in (2) and 3.25 μL of a virus extract having the following composition were added and stirred.

Composition of virus extract: concentration is final concentration after addition of sample and pharyngeal swab (in 15 μL) 21.2 mM magnesium chloride 102.7 mM potassium chloride 1.15% glycerol 11.5% dimethyl sulfoxide 0.01% sodium dodecyl sulfate (4) 10 μL of a reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (GeneAmp Thin-Walled Reaction Tubes, manufactured by Life Technologies). The first primer and the second primer were the oligonucleotides and concentrations of the sequences shown in Table 1, respectively, and the T7 promoter sequence (SEQ ID NO: 8) was added to the 5'end side of the first primer. Further, among the first and second primers shown in Table 1, the primers for amplifying the internal standard nucleic acid and the type A H1N1 subtype influenza virus RNA are the primers having the sequences shown in SEQ ID NOs: 9 and 12, and A The primer for amplifying subtype H3N2 influenza virus is the primer consisting of the sequences shown in SEQ ID NOS: 10 and 13, and the primer for amplifying the influenza B virus consists of the sequence appearing in SEQ ID NOS: 11 and 14. It is a primer.

Composition of reaction solution: final concentration after addition of virus extract and starting solution (in 30 μL) 60 mM Tris-HCl (pH 8.6)
0.25 mM dATP, dCTP, dGTP, dTTP
2.6 mM ATP, CTP, UTP, GTP each
3.06 mM ITP
90 mM trehalose 6.4U AMV reverse transcriptase 142U T7 RNA polymerase 10 ⁴ copies Internal standard (SEQ ID NO: 15)
First primer (see Table 1 for concentration and sequence)
Second primer (see Table 1 for concentration and sequence)

（５）上記反応液を４６℃で４分間保温後、直ちに前記（３）のウイルス抽出液１５μＬ、および以下の組成からなる開始液５μＬを添加し、反応温度４６℃で核酸増幅反応を開始した。

(5) After incubating the above reaction solution at 46 ° C. for 4 minutes, immediately add 15 μL of the virus extract of (3) above and 5 μL of a starting solution having the following composition, and start the nucleic acid amplification reaction at a reaction temperature of 46 ° C. .

Composition of the starting solution: The concentration is the final concentration after addition of the virus extract and the starting solution (in 30 μL) 21.2 mM Magnesium chloride 102.7 mM Potassium chloride 1.2% Glycerol 11.5% Dimethyl sulfoxide 0.5% Tween 20
(6) 8 minutes after the addition of the starting solution, the solution containing the first probe prepared in Example 1 was added, and the solid phase to which the second probe prepared in Example 2 was bound was placed in a PCR tube. Chromatography was carried out for 2 minutes.

FIG. 1 shows the results when the type A H3N2 subtype influenza virus was used as the virus sample, and FIG. 2 shows the results when the type B influenza virus was used. The type A H3N2 subtype influenza virus detected from 5 × 10 ⁻¹ to 5 × 10 ⁻² TCID ₅₀ / mL, and the type B influenza virus detected up to 5 × 10 ⁰ TCID ₅₀ / mL, respectively, type A / B. It can be seen that the influenza virus is detected simply, quickly and with high sensitivity. Further, when the type A H3N2 influenza virus is used as a sample, the position where the oligonucleotide (second probe) having the sequence shown in SEQ ID NO: 5 is immobilized and the position of the internal standard (sequence shown in SEQ ID NO: 7) (The position where the oligonucleotide consisting of is immobilized) (FIG. 1), and when the influenza B virus is used as a sample, the position where the oligonucleotide consisting of the sequence of SEQ ID NO: 6 (the second probe) is immobilized. Since the bands were respectively confirmed only in the positions of (1) and (3) the position of the internal standard (the position where the oligonucleotide consisting of the sequence shown in SEQ ID NO: 7 was immobilized) (FIG. 2), the type of influenza virus It can be seen that discrimination can be performed.

Claims (6)

A method for detecting a virus having an envelope, which comprises the following steps (1) to (3):
(1) a step of contacting a sample containing a virus having an envelope with a solution containing at least a water-soluble organic solvent to extract the nucleic acid derived from the virus;
(2) A primer having a sequence homologous to a part of the specific base sequence in the virus-derived nucleic acid (first primer) and a primer having a sequence complementary to a part of the specific base sequence (second A step of reacting a solution containing a primer) and a nucleic acid amplification enzyme with the nucleic acid extracted in the step (1) to amplify a nucleic acid containing the specific base sequence or a complementary sequence of the specific base sequence;
(3) An oligonucleotide (first probe) bound with a labeling substance capable of hybridizing with the nucleic acid amplified in the step (2) under stringent conditions, and hybridized with the amplified nucleic acid under stringent conditions Detecting the virus-derived nucleic acid by capturing the amplified nucleic acid with an oligonucleotide (second probe) bound to a possible solid phase;
And
The virus with an envelope is influenza virus,
The method wherein the first primer, the second primer, the first probe and the second probe are any combination of the following (i) to (iii).
(I) When the influenza virus is a subtype A H1N1 virus, the first primer is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 9, the second primer is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 12, An oligonucleotide in which one probe has the sequence shown in SEQ ID NO: 1 and the second probe has the sequence shown in SEQ ID NO: 5 (ii) When the influenza virus is a subtype A H3N2 virus, One primer is an oligonucleotide consisting of the sequence described in SEQ ID NO: 10, a second primer is an oligonucleotide consisting of the sequence described in SEQ ID NO: 13, the first probe is an oligonucleotide consisting of the sequence described in SEQ ID NO: 2, and, Origonu the second probe comprises a sequence described in SEQ ID NO: 5 Reotide (iii) When the influenza virus is influenza B virus, the first primer is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 11, the second primer is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 14, Is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 3, and a second probe is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 6. The detection method according to claim 1, wherein the solution containing the water-soluble organic solvent that is brought into contact with the sample containing the virus having an envelope in the step (1) is a solution containing dimethylsulfoxide. The detection method according to claim 2, wherein the solution containing the water-soluble organic solvent that is brought into contact with the sample containing the virus having an envelope in the step (1) is a solution further containing one or more kinds of surfactants. The detection method according to claim 1, wherein the solid phase in the step (3) is a membrane, and the nucleic acid amplified in the step (2) is captured by chromatography using a developing solution. The detection method according to claim 4, wherein the developing solution contains at least a nonionic surfactant. Envelope-containing virus detection reagent kit containing the following reagents (A) to (D):
(A) A reagent for extracting nucleic acid from an enveloped virus, which contains at least dimethyl sulfoxide and one or more kinds of surfactants;
(B) A primer having a sequence homologous to a part of the specific base sequence in the virus-derived nucleic acid (first primer), and a primer having a sequence complementary to a part of the specific base sequence (second A primer) and a reagent for amplifying a nucleic acid containing at least the nucleic acid amplification enzyme and containing the specific base sequence or a complementary sequence of the specific base sequence;
(C) a membrane to which an oligonucleotide (first probe) capable of hybridizing with the amplified nucleic acid under stringent conditions is bound;
(D) an oligonucleotide (second probe) bound with a labeling substance capable of hybridizing with the amplified nucleic acid under stringent conditions;
And the virus having an envelope is an influenza virus,
A kit in which the first primer, the second primer, the first probe, and the second probe are any combination of the following (i) to (iii).
(I) When the influenza virus is a subtype A H1N1 virus, the first primer is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 9, the second primer is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 12, An oligonucleotide in which one probe has the sequence shown in SEQ ID NO: 1 and the second probe has the sequence shown in SEQ ID NO: 5 (ii) When the influenza virus is a subtype A H3N2 virus, One primer is an oligonucleotide consisting of the sequence described in SEQ ID NO: 10, a second primer is an oligonucleotide consisting of the sequence described in SEQ ID NO: 13, the first probe is an oligonucleotide consisting of the sequence described in SEQ ID NO: 2, and, Origonu the second probe comprises a sequence described in SEQ ID NO: 5 Reotide (iii) When the influenza virus is influenza B virus, the first primer is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 11, the second primer is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 14, Is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 3, and a second probe is an oligonucleotide consisting of the sequence shown in SEQ ID NO: 6.

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