Method for extracting viral nucleic acids

Abstract

To provide a method for quickly and simply extracting viral nucleic acid from a sample including a virus.SOLUTION: A sample including virus having no envelope is heated under the presence of solubilizer containing only anionic surfactant, and the viral nucleic acid is extracted.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for extracting a viral nucleic acid from a sample containing a virus. In particular, the present invention relates to a method for rapidly and simply extracting a viral nucleic acid from a sample containing a non-enveloped virus.

Background Art

[0002] In the treatment of genetic diseases, a method of introducing a viral vector containing a gene for the treatment into cells is widely used because of its good introduction efficiency into the cells. However, among the produced viral vectors, there are also vectors that do not contain the gene (so-called empty vectors). Therefore, in order to use the produced viral vector for the treatment of genetic diseases, it is necessary to confirm whether the gene for the treatment exists in the vector and to quantify the gene contained in a sample (such as a cell culture solution) containing the viral vector.

[0003] As methods for qualitatively and / or quantitatively measuring a gene for the treatment of genetic diseases contained in a viral vector, image analysis of viral vector particles by a transmission electron microscope, analytical ultracentrifugation, quantitative PCR (qPCR) method, digital droplet PCR (ddPCR) method, dot blot method, and electrophoresis method are known. Among these, the qPCR method is widely used for the quantification of viral vectors because of its simplicity and rapidity of operation. Further, as nucleic acid amplification methods that are relatively easy to save labor in the reaction process and reduce the cost of the reaction apparatus, NASBA (Nucleic Acid Sequence Based Amplification) method, TMA (Transcription Mediated Amplification) method, and TRC (Transcription Reverse transcription Concerted) method, which can amplify nucleic acids at a constant temperature in a relatively low temperature range (for example, in the range of 40°C to 50°C), are known. These amplification methods are usually amplification methods targeting single-stranded RNA, but DNA amplification is also possible by devising the reaction system (Patent Document 1).

[0004] When detecting genes for the treatment of genetic diseases contained in a viral vector 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 types of surfactants are commonly used as solubilants for viral nucleic acid extraction. Extraction is also often performed using 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).

[0005] However, the conventional methods described above take about 10 minutes to an hour and involve a large number of steps, so there was a need for a faster and simpler method. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2020-162550 [Patent Document 2] Japanese Patent Application Publication No. 8-510004 [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a method for rapidly and easily extracting viral nucleic acid from a sample containing a virus. [Means for solving the problem]

[0008] The inventors selected a virus without an envelope as the target virus for nucleic acid extraction, and after diligently studying the optimal conditions for extracting the nucleic acid of the virus, they completed the present invention.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] The present invention will be described in detail below.

[0014] Examples of non-enveloped viruses that are the target of extraction in this invention include adeno-associated viruses (AAVs), adenoviruses, noroviruses, rotaviruses, papillomaviruses, rhinoviruses, astroviruses, hepatitis A viruses, hepatitis E viruses, and enteroviruses. In particular, the method of this invention is preferred for nucleic acid extraction from AAVs.

[0015] The method of the present invention is characterized in that it contains only an anionic surfactant as a solvent used to dissolve the capsid of a virus without an envelope and extract the viral nucleic acid contained within the capsid. The anionic surfactant can be appropriately selected from among 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, which is 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 more preferably to 0.4% (w / v) or more and 0.6% (w / v) or less. In addition to the anionic surfactant, the solvent may further contain components that do not have the ability to extract viral nucleic acids. Examples of such components include buffer components such as acetate, phosphate, MES (2-Morpholinoethanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Tris (Tris(hydroxymethyl)aminomethane), and borate, as well as substances that can encapsulate substances that inhibit nucleic acid amplification reactions, such as cyclodextrin.

[0016] 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 can be set appropriately, taking into consideration the type of virus (DNA virus or RNA virus) and the concentration of the anionic surfactant contained in the solvent. For example, if the virus without an envelope is a DNA virus such as AAV, and the anionic surfactant is SDS in an amount of 0.003% (w / v) to 1.5% (w / v) or sodium deoxycholate in an amount of 0.3% (w / v) to 0.7% (w / v), it is preferable to set the temperature to 60°C to 98°C, and more preferably to 65°C to 80°C. The heating time can be determined appropriately, taking into consideration the properties of the sample containing the virus, the virus concentration in the sample, the concentration of the solvent (anionic surfactant), etc., but in most cases, viral nucleic acids can be sufficiently extracted by heating for 15 minutes or less.

[0017] 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 solubilizer (anionic surfactant) by ultrafiltration or dialysis, or it may be subjected to a nucleic acid amplification reaction directly or after appropriate dilution. When SDS is used as the anionic surfactant, SDS adversely affects the nucleic acid amplification reaction, so 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 solubilizer should be between 0.003% (w / v) and 0.15% (w / v).

[0018] To amplify a nucleic acid from a solution containing viral nucleic acid obtained by the method of the present invention, the amplification can be carried out using a nucleic acid amplification reagent that 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 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.

[0019] 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, examples of washing conditions include 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 even more preferably 68°C, 0.1×SSC, 0.1% (w / v)SDS.

[0020] When the viral nucleic acid is an AAV vector, a preferred embodiment of the primer set is as follows: <1> from <8> The primer sets shown are examples.

[0021] <1>A primer set in which the first primer is an oligonucleotide that can specifically hybridize with the nucleotide sequence set forth in SEQ ID NO: 1 (the nucleotide sequence from positions 1560 to 1650 of the AAV vector partial sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 3) under stringent conditions, and the second primer is an oligonucleotide that can specifically hybridize with the nucleotide sequence set forth in SEQ ID NO: 2 (the complementary sequence of the nucleotide sequence from positions 1356 to 1516 of the AAV vector partial sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 3) under stringent conditions <2>A primer set in which the first primer is an oligonucleotide that can specifically hybridize with the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 4 (the nucleotide sequence from positions 133 to 150 of the AAV vector partial sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 21) under stringent conditions, and the second primer is an oligonucleotide that can specifically hybridize with the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 5 (from positions 11 to 38 of the AAV vector partial sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 21) under stringent conditions <3>A primer set in which the first primer is an oligonucleotide that can specifically hybridize with the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 6 (the nucleotide sequence from positions 525 to 542 of the AAV vector partial sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 21) under stringent conditions, and the second primer is an oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 7 (from positions 291 to 308 of the AAV vector partial sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 21) or 8 (from positions 201 to 218 of the AAV vector partial sequence consisting of the nucleotide sequence set forth 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 (nucleotide sequences from position 441 to 458 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 the nucleotide sequence described in SEQ ID NO: 10 (nucleotide sequences from position 381 to 398 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 21) or 11 (nucleotide sequences from position 275 to 292 of the AAV vector subsequence 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 (nucleotide sequences 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 (nucleotide sequences 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 (nucleotide sequences 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 one of the nucleotide sequences described in SEQ ID NO: 15 (nucleotide sequences from position 196 to 213 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 21), 16 (nucleotide sequences from position 111 to 128 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 21), and 17 (nucleotide sequences from position 11 to 28 of the AAV vector subsequence consisting of the nucleotide sequence described in SEQ ID NO: 21). <7>A primer set, wherein the first primer is an oligonucleotide capable of specifically hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 18 (a nucleotide sequence from position 189 to position 206 of the AAV vector partial nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 21), and the second primer is an oligonucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 19 (from position 21 to position 38 of the AAV vector partial nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 21).

[0022] <8>A primer set, wherein the first primer is an oligonucleotide capable of specifically hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 20 (a nucleotide sequence from position 105 to position 122 of the AAV vector partial nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 21), and the second primer is an oligonucleotide capable of specifically hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 5.

[0023] A nucleic acid containing a specific nucleotide sequence or a complementary sequence of the specific nucleotide sequence, amplified with a nucleic acid amplification reagent containing the above-described primer set, can detect viral nucleic acid contained in a sample by using a probe capable of specifically hybridizing under stringent conditions with a part of the nucleic acid. As the probe, it is preferable in that amplification and detection of the nucleic acid can be performed in a single step and in a sealed container by using a fluorescent dye-labeled oligonucleotide probe designed to change fluorescence characteristics by hybridizing under stringent conditions with a part of the nucleic acid. Examples of the fluorescent dye-labeled probe include a fluorescent-labeled probe using FRET (fluorescence resonance energy transfer), an oligonucleotide probe labeled with an intercalating fluorescent dye, a TaqMan (trade name) probe, and a Molecular Beacon probe.

Advantages of the Invention

[0024] The present 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. Since the method of the present 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. [Brief explanation of the drawing]

[0025] [Figure 1] 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). [Figure 2] This figure compares the amount of AAV nucleic acid extracted depending on the concentration of the 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. [Examples]

[0026] 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.

[0027] Example 1: Preparation of AAV vectors (1) A nucleotide sequence (SEQ ID NO: 23) was designed by adding the 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 BamHI recognition sequence (GGATTC) to the 3' end.

[0028] (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-EGFP). Escherichia coli strain JM109 was transformed with pUC-EGFP, and the resulting transformants were cultured. pUC-EGFP was extracted from the culture medium using the QIAprep Spin Miniprep kit (Qiagen).

[0029] (3) The pUC-EGFP 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 previously digested with restriction enzymes EcoRI and BamHI. The ligation product was used to transform Escherichia coli strain JM109. The resulting transformants were cultured in LB medium containing 100 μg / mL of carbenicillin, and the EGFP-expressing vector pAAV-EGFP was extracted using the QIAprep Spin Miniprep kit (Qiagen Inc.).

[0030] (4) The transformants obtained in (3) were cultured overnight at 37°C with shaking in a 5L baffled flask containing 1L 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 the culture was complete, the cells were collected by centrifugation. A large amount of pAAV-EGFP was prepared from the collected cells using Plasmid Mega Kit (Qiagen).

[0031] (5) Preparation of AAV2-EGFP (5-1) Escherichia coli strain JM109 was transformed using the pRC2-mi342 Vector (Takara Bio Inc.) and the pHelper Vector (Takara Bio Inc.). Using the resulting transformants, pRC2-mi342 and pHelper were prepared in large quantities by performing the same procedure as in (4).

[0032] (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.

[0033] 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). Benzonase (Merck Millipore) was added at 1 / 2000 volume, 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, 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.

[0034] (5-4) The supernatant from which suspended solids were 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").

[0035] (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.

[0036] (5-6) The AAV2-EGFP concentration in the solution obtained in (5-5) was quantified by qPCR using the AAVpro Titration Kit (Takara Bio Inc.). The solution was then subjected to SDS-PAGE and silver staining using the Pierce Silver Stain Kit (Thermo Fisher Scientific Inc.) to confirm the purity of the AAV vector contained in the solution, and only the bands corresponding to VP1, VP2, and VP3, which constitute the AAV vector, were observed.

[0037] 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.

[0038] [Table 1]

[0039] (2) The treated solution was diluted 250-fold, and the AAV nucleic acid contained in the diluted solution was quantified by qPCR using the 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 Positive Control diluted to various concentrations and the diluted solution were added to 18 μL of the reaction solution having the following composition, and qPCR was performed under the conditions of heating at 25°C for 2 minutes - 53°C for 10 minutes - 95°C for 2 minutes, followed by 40 repetitions of a cycle of 95°C for 3 seconds - 60°C for 30 seconds. The AAV nucleic acid contained in the diluted solution was quantified based on the calibration curve of the Positive Control.

[0040] Composition of reaction solution: Final concentration in 20 μL TaqPath qPCR Master Mix 900nM First primer (SEQ ID NO: 24) 900nM Second primer (SEQ ID NO: 25) 250nM 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 acids were extracted than when using a commercially available extraction reagent (experiment no. 16).

[0041] 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.

[0042] (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 heated at 70°C for 10 minutes.

[0043] (2) After the extraction solution was diluted 250 times, the amount of AAV nucleic acid contained in the diluted solution was quantified using the method described in Example 2(2).

[0044] (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 solubilizing agent, in the same manner as described in (1) and (2).

[0045] 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)), it is possible to extract AAV nucleic acids equivalent to or better than when using commercially available extraction reagents.

[0046] Example 4: Effect of introducing a solvent on nucleic acid amplification reactions In Examples 2 and 3, SDS and sodium deoxycholate were found to be effective solvents for the extraction of AAV nucleic acids. Therefore, the effects of introducing these solvents on the nucleic acid amplification reaction were evaluated.

[0047] (1) Dilute the Positive Control contained in the AAVpro Titration Kit for Real Time PCR (manufactured by Takara Bio) 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 in 1 × 10⁻¹⁶ solutions. 3 The sample was diluted to create a copy / test sample, and this was used as the DNA sample.

[0048] (2) The AAV nucleic acid contained in the DNA sample prepared in (1) was measured by the method described in Example 2(2).

[0049] 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 inhibit the nucleic acid amplification reaction. From Example 3 and these results, it can be seen that when using SDS as a solvent and directly (without dilution) the solution after extraction is used in the nucleic acid amplification reaction, the concentration of SDS should be between 0.003% (w / v) and 0.15% (w / v).

[0050] [Table 2]

Claims

1. 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 sodium deoxycholate in a concentration of 0.3% (w / v) or more and 0.7% (w / v) or less. The aforementioned method.

2. A step of 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 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.

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