JP7830860B2 - Method for extracting viral nucleic acids - Google Patents

Description

This invention relates to a method for extracting viral nucleic acids from a sample containing a virus. In particular, this invention relates to a method for rapidly and easily extracting viral nucleic acids from a sample containing a virus that does not have an envelope.

In the treatment of genetic disorders, the method of introducing the therapeutic gene into cells using a viral vector is widely used due to its high efficiency in cell introduction. However, some manufactured viral vectors do not contain the aforementioned gene (so-called empty vectors). Therefore, in order to use a manufactured viral vector for the treatment of genetic disorders, it is necessary to confirm whether the therapeutic gene is present in the vector and to quantify the amount of the gene present in the sample containing the viral vector (such as cell culture medium).

Methods for qualitatively and/or quantitatively measuring genes for the treatment of genetic diseases contained in viral vectors include image analysis of viral vector particles using transmission electron microscopy, analytical ultracentrifugation, quantitative PCR (qPCR), digital droplet PCR (ddPCR), dot blotting, and electrophoresis. Of these, qPCR is widely used for the quantification of viral vectors due to its ease of operation and speed. Furthermore, as nucleic acid amplification methods that allow for relatively easy reduction of reaction process labor and cost reduction of reaction equipment, the NASBA (Nucleic Acid Sequence-Based Amplification) method, the TMA (Transscription Mediated Amplification) method, and the TRC (Transscription Reverse Translation Concerted) method are known, which enable nucleic acid amplification at a constant temperature of relatively low temperatures (e.g., in the range of 40°C to 50°C). While these amplification methods typically target single-stranded RNA, DNA amplification is also possible by modifying the reaction system (Patent Document 1).

When detecting genes for the treatment of genetic diseases contained in viral vectors using the nucleic acid amplification method described above, it is necessary to extract the genes (viral nucleic acids) from the sample containing the viral vector. Conventionally, combinations of surfactants and proteinase K (Patent Document 2) or combinations of multiple surfactants are commonly used as solubilants for viral nucleic acid extraction. Furthermore, commercially available kits such as EXTRAGEN II (Tosoh Corporation) and RNeasy Mini Kit (Qiagen Corporation), or commercially available instruments such as Chemagic Prepito (PerkinElmer Corporation) are often used for extraction.

However, the conventional methods described above take approximately 10 minutes to an hour and involve a large number of steps, so there was a need for a faster and simpler method.

Japanese Patent Publication No. 2020-162550 Japanese Patent Application Publication No. 8-510004

The object of this invention is to provide a method for rapidly and easily extracting viral nucleic acid from a sample containing a virus.

The inventors selected a virus without an envelope as the target virus for nucleic acid extraction, and after diligently investigating the optimal conditions for extracting the nucleic acid of this virus, they completed the present invention.

In other words, the first aspect of the present invention is
A method for extracting viral nucleic acid, comprising the step of heating a sample containing a virus that does not have an envelope in the presence of a solubilant,
The method comprising only an anionic surfactant as the solvent.

Furthermore, a second aspect of the present invention is the method according to the first aspect, wherein the anionic surfactant is 0.003% (w/v) to 0.15% (w/v) of sodium dodecyl sulfate, or 0.3% (w/v) to 0.7% (w/v) of sodium deoxycholate.

Furthermore, a third aspect of the present invention is:
A step of extracting nucleic acid of a virus without an envelope using the method described in the first or second embodiment,
A step of amplifying the nucleic acid using a nucleic acid amplification reagent that includes a primer set for amplifying the nucleic acid containing the specific base sequence or the complementary sequence of the specific base sequence, comprising a first primer having a sequence complementary to a part of the specific base sequence of the nucleic acid and a second primer having a sequence homologous to a part of the specific base sequence.
The process includes detecting a nucleic acid, which includes a specific base sequence or a complementary sequence of the specific base sequence, amplified in the above step, using a probe that can specifically hybridize with a portion of the nucleic acid under stringent conditions.
This is a method for detecting the aforementioned virus contained in a sample.

Furthermore, a fourth aspect of the present invention is the method according to any of the first to third aspects, wherein the non-enveloped virus is an adeno-associated virus.

The present invention will be described in detail below.

Examples of non-enveloped viruses targeted for extraction in this invention include adeno-associated viruses (AAV), adenoviruses, noroviruses, rotaviruses, papillomaviruses, rhinoviruses, astroviruses, hepatitis A virus, hepatitis E virus, and enteroviruses. The method of this invention is particularly preferred for nucleic acid extraction from AAV.

The method of the present invention is characterized in that the solubilizer used to dissolve the capsid of a virus without an envelope and extract the viral nucleic acid contained within the capsid contains only an anionic surfactant. The anionic surfactant can be appropriately selected from those commonly used as membrane protein solubilizers. Examples include dodecyl sulfate, deoxycholic acid, cholic acid, glycolic acid, taurocholic acid, taurodeoxycholic acid, and their salts. The concentration of the anionic surfactant during viral nucleic acid extraction can be appropriately set considering the critical micelle concentration of the surfactant used and the properties or concentration of impurities contained in the virus-containing sample. For example, when using sodium dodecyl sulfate (SDS), which is a dodecyl sulfate salt, as the anionic surfactant, it is preferable to set the concentration to 0.003% (w/v)% or more and 1.5% (w/v) or less, and more preferably to 0.01% (w/v)% or more and 1% (w/v) or less. Furthermore, when using sodium deoxycholate, a deoxycholate salt, as the anionic surfactant, it is preferable to set the concentration to 0.3% (w/v) or more and 0.7% (w/v) or less, and preferably to 0.4% (w/v) or more and 0.6% (w/v) or less. The solvent may also contain components other than the anionic surfactant that do not have the ability to extract viral nucleic acids. Examples of such components include buffer components such as acetate, phosphate, MES (2-Morphorinoethanesulfonic acid), HEPES (4-(2-hydroxymethyl)-1-piperazineethanesulfonic acid), Tris (Tris(hydroxymethyl)aminomethane), and borate, as well as substances capable of encapsulating substances that inhibit nucleic acid amplification reactions, such as cyclodextrin.

In the method of the present invention, viral nucleic acids are extracted by heating in the presence of a solvent containing only the aforementioned anionic surfactant. The heating temperature should be set appropriately, taking into consideration the type of virus (DNA virus or RNA virus) and the concentration of the anionic surfactant included in the solvent. For example, if the non-enveloped virus is a DNA virus such as AAV, and the anionic surfactant is SDS at 0.003% (w/v) to 1.5% (w/v) or sodium deoxycholate at 0.3% (w/v) to 0.7% (w/v), then setting the temperature to 60°C to 98°C is preferable, and setting it to 65°C to 80°C is more preferable. The heating time should be determined appropriately, taking into consideration the properties of the virus-containing sample, the virus concentration in the sample, the concentration of the solvent (anionic surfactant), etc., but in most cases, heating for 15 minutes or less is sufficient to extract viral nucleic acids.

The solution containing viral nucleic acids extracted by the method of the present invention may be subjected to a nucleic acid amplification reaction after removing the solvent (anionic surfactant) by ultrafiltration or dialysis, or it may be subjected to the nucleic acid amplification reaction directly or after appropriate dilution. When using SDS as the anionic surfactant, since SDS adversely affects the nucleic acid amplification reaction, when the solution containing viral nucleic acids extracted by the method of the present invention is subjected to a nucleic acid amplification reaction directly, the concentration of SDS as a solvent should be between 0.003% (w/v) and 0.15% (w/v).

To amplify a solution containing viral nucleic acid obtained by the method of the present invention, a nucleic acid amplification reagent may be used, which includes a primer set for amplifying nucleic acids containing the specific base sequence or a complementary sequence of the specific base sequence, comprising a first primer having a sequence complementary to a portion of the specific base sequence of the nucleic acid, and a second primer having a sequence homologous to a portion of the specific base sequence.

In this specification, a specific base sequence refers to the base sequence of the viral nucleic acid from the 3' end of the complementary region with the first primer to the 5' end of the homologous region with the second primer. A complementary sequence is a sequence that can specifically hybridize under stringent conditions, and a homologous sequence is a sequence that can specifically hybridize to the complementary sequence of a specific base sequence under stringent conditions. An example of "stringent conditions" here is a condition in which a specific hybrid is formed and a nonspecific hybrid is not formed. To give one example, this is a condition in which polynucleotides with high homology (e.g., identity or similarity), for example, polynucleotides with 70% or more homology, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more homology, hybridize, and polynucleotides with lower homology do not hybridize. While not limited to these, specific hybridization conditions include those at 42°C in the presence of 50% (v/v) formamide, 0.1% (w/v) bovine serum albumin, 0.1% (w/v) Ficol (trade name), 0.1% (w/v) polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, and 75 mM sodium citrate, as well as the nucleic acid amplification conditions described in the examples of this specification. Furthermore, suitable washing conditions include conditions such as washing once, preferably two to three times, at a salt concentration and temperature equivalent to stringent conditions, such as 60°C, 1×SSC (Saline Sodium Citrate Buffer), 0.1% (w/v) SDS, preferably 0.1×SSC, 0.1% (w/v) SDS, more preferably 65°C, 0.1×SSC, 0.1% (w/v) SDS, and more preferably 68°C, 0.1×SSC, 0.1% (w/v) SDS.

When the viral nucleic acid is an AAV vector, preferred embodiments of the primer set include the primer sets shown in <1> to <8> below.

<1> A primer set in which the first primer is an oligonucleotide that can specifically hybridize under stringent conditions to the nucleotide sequence described in SEQ ID NO: 1 (the nucleotide sequence from position 1560 to 1650 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 3), and the second primer is an oligonucleotide that can specifically hybridize under stringent conditions to the nucleotide sequence described in SEQ ID NO: 2 (the complementary sequence of the nucleotide sequence from position 1356 to 1516 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 3). <2> A primer set in which the first primer is an oligonucleotide that can specifically hybridize under stringent conditions to the complementary sequence of the nucleotide sequence described in SEQ ID NO: 4 (the nucleotide sequence from position 133 to 150 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21), and the second primer is an oligonucleotide that can specifically hybridize under stringent conditions to the complementary sequence of the nucleotide sequence described in SEQ ID NO: 5 (the nucleotide sequence from position 11 to 38 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21). <3> A primer set in which the first primer is an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 6 (the nucleotide sequence from position 525 to 542 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21), and the second primer is an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 7 (positions 291 to 308 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21) or 8 (positions 201 to 218 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21). <4> A primer set in which the first primer is an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 9 (the nucleotide sequence from position 441 to 458 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21), and the second primer is an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 10 (from position 381 to 398 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21) or 11 (from position 275 to 292 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21). <5> A primer set in which the first primer is an oligonucleotide that can specifically hybridize under stringent conditions to the complementary sequence of the nucleotide sequence described in SEQ ID NO: 12 (the nucleotide sequence from position 385 to 402 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21), and the second primer is an oligonucleotide that can specifically hybridize under stringent conditions to the complementary sequence of the nucleotide sequence described in SEQ ID NO: 13 (the nucleotide sequence from position 11 to 308 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21). <6> A primer set in which the first primer is an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 14 (the nucleotide sequence from position 301 to 318 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 21), and the second primer is an oligonucleotide consisting of any of the nucleotide sequences described in SEQ ID NO: 15 (positions 196 to 213 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 21), 16 (positions 111 to 128 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 21), and 17 (positions 11 to 28 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 21). <7> A primer set in which the first primer is an oligonucleotide that can specifically hybridize under stringent conditions with the complementary sequence of the nucleotide sequence described in SEQ ID NO: 18 (the nucleotide sequence from position 189 to 206 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21), and the second primer is an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 19 (the nucleotide sequence from position 21 to 38 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21).

<8> A primer set in which the first primer is an oligonucleotide that can specifically hybridize under stringent conditions to the complementary sequence of the nucleotide sequence described in SEQ ID NO: 20 (the nucleotide sequence from position 105 to 122 of the AAV vector partial sequence consisting of the nucleotide sequence described in SEQ ID NO: 21), and the second primer is an oligonucleotide that can specifically hybridize under stringent conditions to the complementary sequence of the nucleotide sequence described in SEQ ID NO: 5.

Nucleic acids containing a specific base sequence or a complementary sequence of the specific base sequence, amplified using the nucleic acid amplification reagent including the primer set described above, can be detected in a sample by using a probe that can specifically hybridize with a portion of the nucleic acid under stringent conditions. It is preferable to use a fluorescent dye-labeled oligonucleotide probe designed to change its fluorescence properties upon hybridization with a portion of the nucleic acid under stringent conditions, as this allows for the amplification and detection of the nucleic acid in a single step and within a sealed container. Examples of such fluorescent dye-labeled probes include FRET (fluorescence resonance energy transfer)-based fluorescently labeled probes, oligonucleotide probes labeled with intercalator fluorescent dyes, TaqMan (trade name) probes, and Molecular Beacon probes.

This invention is characterized by extracting viral nucleic acids from a sample containing a virus without an envelope by heating in the presence of a solvent containing only an anionic surfactant. Because the method of this invention allows for rapid and simple extraction of the nucleic acids, the detection of the nucleic acids using nucleic acid amplification reagents can be performed quickly and easily.

This figure compares the amount of AAV nucleic acid extracted with different solvents and with or without heat treatment. In the figure, the black bars represent the results when heat treatment was performed (70°C for 10 minutes), and the white bars represent the results when no heat treatment was performed (left at room temperature). This figure compares the amount of AAV nucleic acid extracted depending on the concentration of anionic surfactant. (a) shows the results when sodium dodecyl sulfate (SDS) is used as the anionic surfactant, and (b) shows the results when sodium deoxycholate is used.

The following description will be detailed using examples where the non-enveloped virus is an adeno-associated virus (AAV). However, these examples are intended to illustrate one embodiment of the present invention and do not limit it.

Example 1 Preparation of AAV vector (1) A nucleotide sequence (SEQ ID NO: 23) was designed by adding a restriction enzyme EcoRI recognition sequence (GAATTC) to the 5' end of a polynucleotide encoding EGFP (Enhanced green fluorescent protein) consisting of the amino acid sequence described in SEQ ID NO: 22, and a stop codon (TAG) and a BamHI recognition sequence (GGATTC) to the 3' end.

(2) A polynucleotide consisting of the sequence described in Sequence ID No. 23 was totally synthesized and cloned into a plasmid (commissioned to FASMAC, named pUC-EGGFP). E. coli strain JM109 was transformed with pUC-EGGFP, and the resulting transformants were cultured. pUC-EGGFP was extracted from the culture medium using the QIAprep Spin Miniprep kit (Qiagen).

(3) The pUC-EGGFP obtained in (2) was digested with restriction enzymes EcoRI and BamHI, and then ligated to the expression vector pAAV-CMV (Takara Bio Inc.), which had been pre-digested with restriction enzymes EcoRI and BamHI. E. coli strain JM109 was transformed using this ligation product. The resulting transformants were cultured in LB medium containing 100 μg/mL carbenicillin, and the EGFP-expressing vector pAAV-EGGFP was extracted using the QIAprep Spin Miniprep kit (Qiagen Inc.).

(4) The transformants obtained in (3) were cultured overnight at 37°C in a 5 L baffled flask containing 1 L of 2YT medium (1.6% (w/v) Tryptone, 1% (w/v) Yeast Extract, 0.5% (w/v) sodium chloride) with 100 μg/mL of carbenicillin. After culturing, the cells were collected by centrifugation. Large quantities of pAAV-EGFP were prepared from the collected cells using Plasmid Mega Kit (Qiagen).

(5) Preparation of AAV2-EGFP (5-1) Escherichia coli strain JM109 was transformed using pRC2-mi342 Vector (Takara Bio Inc.) and pHelper Vector (Takara Bio Inc.). pRC2-mi342 and pHelper were prepared in large quantities by performing the same procedure as in (4) using the transformed cells.

(5-2) HEK293T cells were cultured in 10 T-225 flasks (Thermo Fisher Scientific) containing 45 mL of D-MEM (Dulbecco’s Modified Eagle’s Medium, Fujifilm Wako Pure Chemical Industries) with 10% (v/v) bovine serum. Gene transduction was performed by adding a complex of pAAV-EGFP prepared in (4), pRC2-mi342 and pHelper prepared in (5-1), and polyethyleneimine (Polysciences), and the cells were cultured statically for 3 days under conditions of 5% (v/v) carbon dioxide and 37°C. After culturing, the cells were harvested after being detached by centrifugation, and each cell obtained from 5 T-225 flasks was frozen and stored at -80°C.

The frozen cells obtained in (5-3) and (5-2) were thawed and suspended in 10 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride, 4 mM magnesium chloride, and 0.01% (w/v) Tween 20 (trade name). 1/2000 volume of Benzonase (Merck Millipore) was added, and the mixture was allowed to stand at 37°C for 1 hour. The mixture was then centrifuged at 13000 × g at 4°C for 10 minutes to obtain the supernatant. Ammonium sulfate was added to the supernatant to a 15% saturation point, and the mixture was centrifuged again under the same conditions. The supernatant was passed through a 0.45 μm pore size filter to remove suspended solids.

(5-4) The supernatant, from which suspended solids had been removed, was applied to a 5 mL AVB Sepharose column (manufactured by Cytiva) that had been pre-equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride (hereinafter also referred to as "equilibrium solution A").

(5-5) After washing with equilibration solution A, the cells were eluted with 0.1 M acetate buffer (pH 2.5) containing 0.5 M sodium chloride. The resulting eluate was neutralized by adding 1/4 volume of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride to obtain the AAV2-EGFP solution, which is the AAV vector.

The AAV2-EGFP concentration in the solutions obtained in (5-5) and (5-6) was quantified by qPCR using the AAVpro Titration Kit (Takara Bio Inc.). The solutions were then subjected to SDS-PAGE, and the purity of the AAV vector contained in the solutions was confirmed by silver staining using the Pierce Silver Stain Kit (Thermo Fisher Scientific). Only the bands corresponding to VP1, VP2, and VP3, which constitute the AAV vector, were observed.

Example 2: Nucleic acid extraction from AAV vectors (Part 1)
(1) The AAV2-EGFP solution obtained in Example 1 was treated at 70°C (with heat treatment) or room temperature (without heat treatment) for 10 minutes in the presence of one of the solvents shown in Table 1.

(2) The treated solution was diluted 250-fold, and the AAV nucleic acid contained in the diluted solution was quantified by qPCR using Positive Control contained in the AAVpro Titration Kit for Real Time PCR (manufactured by Takara Bio) as the standard concentration. Specifically, 2 μL of the aforementioned Positive Control diluted to various concentrations and the diluted solution were added to 18 μL of the reaction solution having the following composition. QPCR was performed under the following conditions: heating at 25°C for 2 minutes, 53°C for 10 minutes, and 95°C for 2 minutes, followed by 40 repetitions of a cycle of 95°C for 3 seconds and 60°C for 30 seconds. The AAV nucleic acid contained in the diluted solution was quantified based on the calibration curve of the aforementioned Positive Control.

Reaction solution composition: Final concentration in 20 μL TaqPath qPCR Master Mix
900 nM First primer (SEQ ID NO: 24)
900 nM Second primer (SEQ ID NO: 25)
250 nM TaqMan probe (SEQ ID NO: 26)
The results are shown in Figure 1. Note that the extraction volume [copies/μL] shown in Figure 1 is the value converted to the concentration of the AAV2-EGFP solution before dilution in (2). By using only an anionic surfactant, SDS (experiment no. 4) or sodium deoxycholate (experiment no. 5), as a solvent and applying heat treatment (70°C for 10 minutes), more AAV nucleic acid was extracted than when using a commercially available extraction reagent (experiment no. 16).

Example 3: Nucleic acid extraction from AAV vectors (Part 2)
Based on Example 2, solvents containing only the anionic surfactant SDS (Experiment No. 4) or sodium deoxycholate (Experiment No. 5) yielded favorable results, so the optimal concentrations of these anionic surfactants were investigated.

(1) AAV nucleic acid was extracted in the same manner as in Example 2(1), except that a 0.001% (w/v), 0.01% (w/v), 0.1% (w/v), 0.2% (w/v), 0.5% (w/v), or 1% (w/v) SDS or sodium deoxycholate was used as a solvent and the mixture was heat-treated at 70°C for 10 minutes.

(2) After diluting the solution following the extraction process 250 times, the amount of AAV nucleic acid contained in the diluted solution was quantified using the method described in Example 2(2).

(3) As a control, AAV nucleic acid was extracted and quantified using a commercially available extraction reagent (Lysis buffer, manufactured by Takara Bio Inc.) as a solubilizer, in the same manner as described in (1) and (2).

The results are shown in Figure 2. Note that the extraction volume [copies/μL] shown in Figure 2 is the value converted to the concentration of the AAV nucleic acid extract before dilution in (2). It can be seen that under conditions of 0.003% (w/v) to 1.5% (w/v) for SDS (Figure 2(a)) and 0.3% (w/v) to 0.7% (w/v) for sodium deoxycholate (Figure 2(b)), the same or better results can be obtained as when using commercially available extraction reagents.

Example 4: Effect of introducing a solvent on the nucleic acid amplification reaction. In Examples 2 and 3, SDS and sodium deoxycholate were found to be effective solvents for the extraction of AAV nucleic acids. The effect of introducing these solvents on the nucleic acid amplification reaction was evaluated.

(1) Positive Control contained in the AAVpro Titration Kit for Real Time PCR (manufactured by Takara Bio) was diluted with 0.001% (w/v), 0.01% (w/v), 0.1% (w/v), 0.2% (w/v), 0.5% (w/v), or 1% (w/v) SDS or sodium deoxycholate to a concentration of 1 × ^10³ copies/test, and this was used as the DNA sample.

(2) The AAV nucleic acid contained in the DNA sample prepared in (1) was measured using the method described in Example 2(2).

The results are shown in Table 2. SDS was undetectable at concentrations of 0.2% (w/v) or higher, and sodium deoxycholate was undetectable at concentrations of 1% (w/v) or higher, indicating that they inhibited the nucleic acid amplification reaction. From Example 3 and these results, it can be concluded that when using SDS as a solvent and directly (without dilution) the extracted solution for the nucleic acid amplification reaction, the SDS concentration should be between 0.003% (w/v) and 0.15% (w/v).

Claims (2)

A step of extracting viral nucleic acid, which includes a step of heating a sample containing a virus that does not have an envelope in the presence of a solvent,
A step of amplifying the viral nucleic acid using a nucleic acid amplification reagent that includes a primer set for amplifying the viral nucleic acid containing the specific base sequence or a complementary sequence of the specific base sequence, comprising a first primer consisting of a nucleotide sequence described in SEQ ID NO: 24 having a sequence complementary to a part of the specific base sequence of the viral nucleic acid, and a second primer consisting of a nucleotide sequence described in SEQ ID NO: 25 having a sequence homologous to a part of the specific base sequence.
A step of detecting nucleic acid, which includes a specific base sequence or a complementary sequence of the specific base sequence, amplified in the above step, using a probe consisting of a nucleotide sequence described in Sequence ID No. 26 that can specifically hybridize with a portion of the viral nucleic acid under stringent conditions;
including,
A method for detecting the virus contained in a sample,
The aforementioned virus that does not have an envelope is an adeno-associated virus.
The aforementioned dissolving agent comprises only an anionic surfactant.
The anionic surfactant is sodium deoxycholate in a concentration of 0.3% (w/v) or more and 0.7% (w/v) or less.
The aforementioned method. A step of extracting viral nucleic acid, which includes a step of heating a sample containing a virus that does not have an envelope in the presence of a solvent,
A step of amplifying the viral nucleic acid using a nucleic acid amplification reagent that includes a primer set for amplifying the viral nucleic acid containing the specific base sequence or a complementary sequence of the specific base sequence, comprising a first primer consisting of a nucleotide sequence described in SEQ ID NO: 24 having a sequence complementary to a part of the specific base sequence of the viral nucleic acid, and a second primer consisting of a nucleotide sequence described in SEQ ID NO: 25 having a sequence homologous to a part of the specific base sequence.
A step of detecting the nucleic acid, which includes a specific base sequence or a complementary sequence of the specific base sequence, amplified in the above step, using a probe consisting of a nucleotide sequence described in Sequence ID No. 26 that can specifically hybridize with a part of the viral nucleic acid under stringent conditions,
including,
A method for detecting the virus contained in a sample,
The aforementioned virus that does not have an envelope is an adeno-associated virus.
The aforementioned dissolving agent comprises only an anionic surfactant.
The anionic surfactant is 0.003% (w/v) to 0.15% (w/v) of sodium dodecyl sulfate.
The aforementioned method.