JP6801237B2 - Nucleic acid extraction and amplification reagents from viruses - Google Patents

Description

The present invention relates to a reagent for extracting and amplifying nucleic acid from a virus easily, quickly and with high efficiency, and a nucleic acid extraction and amplification method using the reagent.

In the field of clinical examination of infectious diseases, it is important to detect pathogens easily, quickly and with high sensitivity for the purpose of obtaining accurate test results and providing appropriate treatment during outpatient treatment. For example, in testing for influenza virus infections that repeat epidemics every winter, rapid test kits are widely used because there are therapeutic agents that are highly effective when administered in the early stages of infection.

As the principle of such a rapid inspection method, the detection method by the immunochromatography method is most used. However, since the detection method by the immunochromatography method has low detection sensitivity, there is a problem that it cannot be detected when the number of pathogens in the sample is small such as in the early stage of infection.

As a method for detecting a pathogen with higher sensitivity than the immunochromatography method, RT-PCR method, TMA method (Patent Document 1), TRC method (Patent Document 2, Non-Patent Document 1), NASBA method (Patent Documents 3 and 4), etc. A detection method using the nucleic acid amplification method of is known. Since the detection method using the nucleic acid amplification method specifically amplifies and detects the nucleic acid possessed by the pathogen, there is a possibility that the pathogen can be detected quickly and with high sensitivity.

When detecting a pathogen by a detection method using a nucleic acid amplification method, generally, an operation of extracting nucleic acid from a sample containing the pathogen is performed in advance. As a method for extracting nucleic acid from a pathogen, particularly a virus, a method using a protein denaturing agent such as a surfactant or guanidine or an organic solvent such as phenol or chloroform is known (Patent Documents 5 and 6). However, since these protein denaturants and organic solvents inhibit the nucleic acid amplification reaction, a purification step for removing them is required after the extraction step, which requires a complicated and time-consuming operation.

Various kits and devices are commercially available to simplify such nucleic acid extraction and purification operations. As an example, a kit using a reagent that insolubilizes nucleic acids such as EXTRAGEN II (manufactured by Tosoh) and centrifugation, a kit using a spin column such as QIAamp Viral RNA Mini Kit (manufactured by Qiagen), and magnetism such as Chemagic Prepito (manufactured by PerkinElmer) An automatic extraction device using beads and the like can be mentioned. However, even if the above-mentioned kit or device is used, it takes about 10 minutes to 1 hour, the work man-hours are large, and a special device such as a centrifuge or an automatic extraction device is required, so that it is quicker and simpler. A method was sought.

On the other hand, there is also known a method of detecting a target nucleic acid by amplifying the nucleic acid without extracting and purifying the nucleic acid as in the method described in Non-Patent Document 2, but the sensitivity is insufficient. It was.

As a method of performing only a nucleic acid extraction operation and subjecting it to a nucleic acid amplification reaction without purification, after extracting nucleic acid with a surfactant, the surfactant is neutralized with cyclodextrin and subjected to a nucleic acid amplification step. The method is known. In Patent Document 7, nucleic acid is extracted from yeast and Escherichia coli using 0.5% sodium dodecyl sulfate (SDS), and nucleic acid is amplified by the PCR method or the RCA method using a nucleic acid amplification reagent containing α-cyclodextrin. The method is shown. However, the method described in the patent document requires heating at 95 ° C. for 5 to 15 minutes at the time of nucleic acid extraction, which is not preferable in terms of convenience, speed, safety and the like. Further, the patent document also shows that yeast-derived genomic DNA can be nucleic acid-amplified by a PCR reaction solution containing cyclodextrin even under the condition that a surfactant other than SDS is added. However, except when the SDS is used, no example is shown in which nucleic acid is extracted from cells or viruses by a surfactant.

Further, Patent Document 8 shows an example in which a nasal swab derived from an influenza virus-infected patient is suspended in a solution containing 0.2% SDS, mixed with an enzyme reagent containing cyclodextrin, and detected by the RT-LAMP method. ing. However, in the patent document, the description about the detection sensitivity and the extraction efficiency was not recognized, and the reagent for extracting and amplifying the nucleic acid from the virus with high efficiency was not known.

Further, the nucleic acid amplification methods of Patent Documents 7 and 8 are methods of amplifying DNA at a relatively high temperature by using a DNA-dependent DNA polymerase, and nucleic acid amplification by a concerted reaction of RNA polymerase and reverse transcriptase at a relatively low temperature. Nucleic acid extraction and amplification conditions from the virus in the method of performing the above (TMA method, TRC method, NASBA method, etc.) were not known.

Japanese Patent No. 2650159 Japanese Patent No. 3152927 Japanese Patent No. 3241717 Japanese Patent No. 4438110 Japanese Unexamined Patent Publication No. 2011-115122 Japanese Patent No. 4303239 Japanese Unexamined Patent Publication No. 2012-10666 Japanese Unexamined Patent Publication No. 2014-198029

Ishiguro, T.I. et al, Analytical Biochemistry, 314, 77-86 (2003) Takeshi Matsumura, The University of Tokyo Dissertation, Report No. 122590 (2007)

An object of the present invention is to provide a nucleic acid extraction and amplification reagent characterized by easily, rapidly and highly efficiently extracting nucleic acid from a virus and minimizing inhibition of a nucleic acid amplification reaction.

As a result of diligent studies to solve the above problems, the present inventors have brought the virus into contact with an extraction reagent containing at least a surfactant having a steroid skeleton, and then further contacted with cyclodextrin for complicated extraction. They have found that nucleic acids can be extracted and amplified with high efficiency without performing and / or purification operations, and have completed the present invention.

That is, the present invention includes the following aspects:
[1] A kit for extracting and amplifying the viral nucleic acid from a sample containing a virus, which comprises the following (i), (ii) and (iii).
(I) Viral nucleic acid extraction reagent containing at least a surfactant having a steroid skeleton (ii) Cyclodextrin (iii) Nucleic acid amplification reagent [2] The cyclodextrin is contained in the nucleic acid amplification reagent, according to [1]. A kit for extracting and amplifying viral nucleic acids.
[3] A kit for extracting and amplifying the viral nucleic acid according to [1] or [2], wherein the virus is an enveloped virus.
[4] A kit for extracting and amplifying the viral nucleic acid according to any one of [1] to [3], wherein the virus is an influenza virus.
[5] A kit for extracting and amplifying the viral nucleic acid according to any one of [1] to [4], wherein the cyclodextrin is γ-cyclodextrin.
[6] A kit for extracting and amplifying the viral nucleic acid according to any one of [1] to [5], wherein the surfactant is a bile acid.
[7] The kit for extracting and amplifying the viral nucleic acid according to [6], wherein the surfactant is either cholic acid, taurocholic acid, glycocholic acid, tauroursodeoxycholic acid or a salt thereof.
[8] A kit for extracting and amplifying the viral nucleic acid according to any one of [1] to [7], wherein the concentration of cyclodextrin is 3 times or more the concentration of the surfactant having a steroid skeleton. ..
[9] Extraction and amplification of the viral nucleic acid according to any one of [1] to [8], wherein the concentration of the surfactant having a steroid skeleton in the extraction reagent is 0.4 (w / w)% or more. Kit to do.
[10] A kit for extracting and amplifying the viral nucleic acid according to any one of [1] to [9], which further comprises a nonionic surfactant.
[11] A kit for extracting and amplifying the viral nucleic acid according to any one of [1] to [10], wherein the nucleic acid amplification reagent is in a dry form.
[12] A method for extracting and amplifying the viral nucleic acid from a sample containing a virus, which comprises the following steps (i), (ii) and (iii).
(I) Nucleic acid extraction step (ii) of contacting the virus with a viral nucleic acid extraction reagent containing at least a surfactant having a steroid skeleton A step of contacting cyclodextrin with the viral nucleic acid extract extracted by (i) (iii). ) Nucleic acid amplification step of contacting a part or all of the solution obtained in the step (ii) with a nucleic acid amplification reagent [13] The viral nucleic acid from a sample containing a virus, which comprises the following steps (i) and (ii). How to extract and amplify.
(I) A nucleic acid amplification reagent containing cyclodextrin from a viral nucleic acid extract extracted by the nucleic acid extraction steps (ii) and (i) in which the virus is brought into contact with a viral nucleic acid extraction reagent containing at least a surfactant having a steroid skeleton. Nucleic acid amplification step in contact with.

Hereinafter, the present invention will be described in detail.

In the present invention, the surfactant having a steroid skeleton is a compound having a cyclopentanoperhydrophenantrene structure and having a surfactant action. Bile acids are exemplified as surfactants having a naturally occurring steroid skeleton. Bile acids are steroid derivatives with a colanic acid skeleton that often combine with glycine and taurine to form conjugated bile acids. Examples of naturally occurring bile acids and bile acid derivatives include cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, tauroursodeoxycholic acid, taurocholic acid, glycocholic acid and salts thereof. The salt is not particularly limited, and examples thereof include sodium salts. Further, the surfactant in the present invention is not limited to a naturally occurring steroid derivative or a bile acid derivative, and may be a surfactant having an artificially synthesized steroid skeleton or colanic acid skeleton. Among them, cholic acid, taurocholic acid, glycocholic acid, tauroursodeoxycholic acid and salts thereof are exemplified as suitable surfactants in the present invention, and cholic acid and salts thereof, particularly sodium cholic acid are exemplified. The concentration of the surfactant having a cholic acid skeleton is not particularly limited, but is preferably 0.4 (w / w)% or more, more preferably 0.8 (w / w)% or more.

The extraction target of the present invention may be any type of virus. Viruses have a structure in which nucleic acids are encapsulated by a protein shell called a capsid, and are clearly distinguished from bacteria, microorganisms, cells, etc. in terms of structure and size. As an example of a preferable extraction target in the present invention, a virus having an envelope and a virus having a single-strand RNA are exemplified. The enveloped virus is a virus in which the virus capsid is covered with an envelope mainly composed of lipids, and examples thereof include simple herpes virus, influenza virus, RS virus, and AIDS virus (HIV). Viruses with single-strand RNA belong to Group 4 (single-strand RNA + strand), Group 5 (positive-strand RNA-strand), and Group 6 (positive-strand RNA reverse transcription) in the Baltimore classification. It is a virus, and examples thereof include corona virus, RS virus, human metapneumovirus, influenza virus, and AIDS virus (HIV). A virus having a single-stranded RNA is preferable because it is suitable for a nucleic acid amplification method using RNA as a starting material, such as the TRC method. Among them, influenza virus is particularly preferable as the virus to be extracted in the extraction reagent of the present invention.

In the present invention, the sample containing the virus includes a sample obtained by simply dissolving the virus in water, physiological saline or a buffer solution, a biological sample containing the virus itself, a filter paper containing the biological sample, a swab and the like. Examples of the biological sample include blood, feces, urine, sputum, lymph, plasma, ejaculation, pulmonary aspirate, cerebrospinal fluid, pharyngeal swab, nasal swab, mouthwash, saliva, and tears.

The viral nucleic acid extraction reagent of the present invention may further contain various components in addition to the surfactant having a steroid skeleton. Containing components necessary for the subsequent nucleic acid amplification reaction, such as enzymes, salts such as magnesium salt and potassium salt, sugars such as trehalose, sugar alcohols such as glycerol, nucleic acid components, organic solvents, etc., is a nucleic acid. It is preferable in the sense that the operation from the extraction step to the nucleic acid amplification step can be simplified. Among them, the organic solvent is particularly preferable because it has an effect of promoting the extraction of viral nucleic acids. Dimethyl sulfoxide (DMSO) can be exemplified as an organic solvent further contained in the extraction reagent of the present invention. Further, in addition to the surfactant having a steroid skeleton, one or more kinds of surfactants and protein denaturing agents can be added as long as they do not interfere with the nucleic acid amplification reaction. As an example of the surfactant to be added, an anionic surfactant such as SDS, an amphoteric surfactant such as CHAPS, Span 20 (trade name), MEGA-8 (trade name), and polyoxyethylene sorbitan fatty acid ester. Nonionic surfactants such as (Tween 20 (trade name), Tween 40 (trade name), Tween 60 (trade name), Tween 80 (trade name), etc.) can be mentioned. Of these, nonionic surfactants are preferable because they have the effect of promoting the extraction of viral nucleic acids and have little effect on the nucleic acid amplification reaction. As an example of the surfactant to be added, Tween 20 (trade name), which is a polyoxyethylene sorbitan fatty acid ester-based surfactant, can be exemplified.

In order to extract the virus nucleic acid from the sample containing a virus by using the virus nucleic acid extraction reagent of the present invention, it is sufficient to simply bring the virus nucleic acid extraction reagent of the present invention into contact with the sample containing the virus. The contact time may be appropriately determined in consideration of the properties of the sample containing the virus, the virus concentration in the sample, the components contained in the virus nucleic acid extraction reagent, etc., but in most cases, the virus nucleic acid is sufficiently obtained within 4 minutes after the contact. It can be extracted and can be subjected to a post-process such as a step of contacting with cyclodextrin immediately after contact.

The virus nucleic acid extract of the present invention can be obtained by using a sample containing a virus and a virus nucleic acid extraction reagent. In addition, when the viral nucleic acid extract comes into contact with cyclodextrin, the surfactant having a steroid skeleton in the viral nucleic acid extract is included in the cyclodextrin, and the inhibitory action on the nucleic acid amplification reaction is alleviated. Cyclodextrins having different ring sizes such as α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, and various derivatives are known. The cyclodextrin used in the present invention may be of any type as long as it can encapsulate a surfactant having a steroid skeleton, but γ-cyclodextrin and its derivatives are preferably used from the viewpoint of ring size. Further, the cyclodextrin to be brought into contact with the virus nucleic acid extract may have any property such as a solution or a solid. Furthermore, various other components may be added at the same time as cyclodextrin.

Cyclodextrin may be contacted between the virus nucleic acid extraction reagent and the sample containing the virus and before the start of the nucleic acid amplification step, and should be appropriately selected within the above range according to the required extraction efficiency. Can be done. In particular, as an example of a preferred embodiment of the present invention, a method in which contact with cyclodextrin is performed at the same time as the start of the nucleic acid amplification step can be exemplified. Further, since it is more preferable that cyclodextrin is added at the same time as the components required for the nucleic acid amplification reagent from the viewpoint of simplifying the operation, a nucleic acid amplification reagent containing cyclodextrin can also be used.

In particular, components necessary for nucleic acid amplification reactions, such as enzymes, salts such as magnesium salts and potassium salts, sugars such as trehalose, sugar alcohols such as glycerol, nucleic acid components, organic solvents, and nucleic acids such as primers and probes. It is preferable to include a type, a buffer solution component, and the like because the operation can be simplified. That is, as an example of a preferred embodiment of the present invention, a viral nucleic acid is extracted with a viral nucleic acid extraction reagent containing a surfactant having a steroid skeleton, and the viral nucleic acid extract is added to a nucleic acid amplification reagent containing cyclodextrin. Examples of the reagent that carries out a nucleic acid amplification reaction in. The concentration of cyclodextrin is not particularly limited, but is preferably 3 times or more, more preferably 6 times or more the concentration of the surfactant having a cholic acid skeleton.

The nucleic acid amplification method in the present invention may be any nucleic acid amplification method (for example, PCR method, TMA method, TRC method, NASBA method). The method of performing nucleic acid amplification at an isothermal temperature is preferable in that a relatively complicated device for raising and lowering the temperature is not required. As such a nucleic acid amplification method, a first primer having a sequence homologous to a part of viral nucleic acid, a second primer having a sequence complementary to a part of viral nucleic acid, and RNA-dependent DNA polymerase activity A method of amplifying a target nucleic acid by using an enzyme having a DNA-dependent DNA polymerase activity, an enzyme having a ribonuclease H (RNase H) activity, and an enzyme having an RNA polymerase activity. A method in which a promoter sequence corresponding to the enzyme having RNA polymerase activity is added to either the first primer or the second primer on the 5'terminal side thereof (for example, TMA method, TRC method, NASBA). Law) is exemplified. The method is a method of amplifying nucleic acid targeting single-stranded RNA, but double-stranded DNA or the like can also be amplified by using the method disclosed in JP-A-2015-11636.

The nucleic acid amplification reagent in the present invention may be in a dry form. Since the nucleic acid amplification reagent contains an enzyme, the storage period can be extended by drying the nucleic acid amplification reagent, and the temperature suitable for storage can be brought closer to room temperature than in the liquid state. Furthermore, there is an advantage that it is easy to operate when transporting or using it by making it dry.

Any method may be used as long as the method does not lose the activity of the enzyme, and examples thereof include a freeze-drying method and an evaporation-drying method.

The present invention extracts and amplifies a viral nucleic acid, which comprises extracting the viral nucleic acid from a sample containing a virus with a viral nucleic acid extracting reagent containing at least a surfactant having a steroid skeleton, and further contacting the viral nucleic acid with cyclodextrin. This is an invention relating to a reagent for virus. According to the present invention, the extraction and amplification of the viral nucleic acid can be easily, quickly and highly efficiently performed without performing a purification operation.

Specifically, a kit and method for extracting and amplifying the viral nucleic acid from a sample containing a virus that adopts the configuration of the present application eliminates the need for complicated extraction steps and has the same detection sensitivity as QIAamp Viral RNA Mini. Was able to be achieved.

Hereinafter, the present invention will be described in detail with reference to Examples and Reference Examples when an influenza virus is used as the virus, but the present invention is not limited to these examples.

Example 1 Preparation of standard RNA The influenza virus RNA (hereinafter referred to as standard RNA) used in the examples described later was prepared by the method shown below.
(1) SEQ ID NO: 1 (A type (H1N1 subtype) influenza virus, segment 5, cDNA partial sequence, GenBank No. FJ996536 from 410th to 930th), SEQ ID NO: 2 (A type (H3N2 subtype) influenza virus , Segment 5, cDNA partial sequence, GenBank No. NC_007369 from 455th to 975th (however, 663rd C is T)) and SEQ ID NO: 3 (Influenza B virus, segment 7, cDNA partial sequence, GenBank No. CY115184). Double-stranded DNA consisting of the nucleotide sequences described in (206th to 735th) was prepared and inserted into T-Vector pMD20 (manufactured by Takara Bio) (note that the 5'end side of the complementary strand of the cDNA sequence). SP6 promoter is added to).
(2) The plasmid DNA prepared in (1) was digested with a restriction enzyme on the 5'end side of the inserted influenza virus cDNA to prepare a linear DNA.
(3) Using the DNA prepared in (2) as a template, in vitro transcription with SP6 RNA polymerase was performed. Then, the template DNA was completely digested by DNase I treatment and purified to prepare standard RNA. The prepared RNA was quantified by measuring the absorbance at 260 nm.

The total length of the standard RNA prepared in this example is about 500 bases, which is a part of the total length of influenza virus RNA (segment 5: about 1500 bases, segment 7: about 1100 bases), but for measurement of influenza virus RNA. Is fully applicable.

Example 2 Preparation of Intercalatorable Fluorescent Dye-labeled Nucleic Acid Probe According to the method of Ishiguro et al. (Ishiguro, T. et al, Nucleic Acids Res., 24, 4992-4997 (1996)), the sequence shown in SEQ ID NO: 4 (GenBank). Between the 17th T and the 18th T from the 5'end of No. KJ741989 (complementary sequence of the base sequence from 724th to 746th), and the sequence shown in SEQ ID NO: 5 (genbank No. CY115184, 463rd Oxazole Yellow is bound to the phosphate diester moiety between the 5'end to the 8th A and the 9th T via a linker (complementary sequence of the base sequence from 483) to labeled with an intercalating fluorescent dye. The nucleic acid probe (hereinafter referred to as INAF probe) was prepared. The structural formula is shown below.

式中、Ｂ^１、Ｂ^２、Ｂ^３、Ｂ^４は塩基を示す。なお、３’末端側−ＯＨからの伸長反応を防止するために３’末端側−ＯＨはグリコール酸修飾がなされている。

In the formula, B ¹ , B ² , B ³ , and B ⁴ indicate bases. The 3'-terminal side-OH is glycolic acid-modified in order to prevent an extension reaction from the 3'-terminal side-OH.

Reference Example 1 Detection of influenza virus by nucleic acid extraction using a spin column We attempted to detect influenza virus by nucleic acid extraction using a spin column by the following method.
(1) The influenza virus solution (manufactured by ZeptoMetrix) shown in Table 1 is diluted to the concentration shown in Table 3 using water for injection, and extracted using QIAamp Virus RNA Mini Kit (manufactured by QIAGEN) according to the manual of the kit. By doing so, influenza virus RNA was obtained.

（２）（１）で調製したインフルエンザウイルスＲＮＡ ２．５μＬを、予め４６℃に加熱した以下の組成からなる溶液 １２．５μＬに添加、撹拌し、４６℃で４分間保温した。

(2) 2.5 μL of influenza virus RNA prepared in (1) was added to 12.5 μL of a solution having the following composition preheated to 46 ° C., stirred, and kept warm at 46 ° C. for 4 minutes.

Composition of virus extract: Concentration is final concentration 42.4 mM magnesium chloride 205.4 mM potassium chloride 2.3% glycerol 23.0% DMSO after addition of virus sample (in 15 μL)

（３）以下の組成からなる反応液１５μＬを０．５ｍＬ容量ＰＣＲチューブ（Ｉｎｄｉｖｉｄｕａｌ Ｄｏｍｅ Ｃａｐ ＰＣＲ Ｔｕｂｅ、ＳＳＩ製）に分注し、４６℃で４分間保温後、（２）の溶液１５μＬを直ちに添加した。なお、第一のプライマーおよび第二のプライマーには、表２に示す配列および濃度からなるオリゴヌクレオチドの組み合わせを用いた。また第一のプライマーには、表２に示した各配列番号に記載の塩基配列の５’末端側に、Ｔ７プロモーター配列（配列番号６）が付加されている。

(3) Dispense 15 μL of the reaction solution having the following composition into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), incubate at 46 ° C. for 4 minutes, and then immediately add 15 μL of the solution of (2). did. For the first primer and the second primer, a combination of oligonucleotides having the sequences and concentrations shown in Table 2 was used. Further, in the first primer, a T7 promoter sequence (SEQ ID NO: 6) is added to the 5'end side of the nucleotide sequence shown in each SEQ ID NO: shown in Table 2.

Reaction solution composition: Concentration is the final concentration 60 mM Tris-HCl (pH 8.6) after addition of viral RNA extract (in 30 μL).
0.25 mM dATP, dCTP, dGTP, dTTP, respectively
2.6 mM ATP, CTP, UTP, GTP respectively
3.06 mM ITP
70 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase 20 nM each INAF probe (prepared in Example 2) (SEQ ID NOs: 4 and 5)
First primer with the concentration shown in Table 2 Second primer with the concentration shown in Table 2.

(4) Continue to use a fluorescence spectrophotometer with a temperature control function (TRCRapid-160, manufactured by Tosoh) that can directly measure the PCR tube, and react at 46 ° C. At the same time, the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm). ) Was measured over time for 30 minutes. A positive determination was made when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.2, with the addition of the starting solution as 0 minutes.

The results are shown in Table 3. The nucleic acid extraction method using the spin column and generally sure it can detect 4.7 × ¹⁰ -2 _TCID 50 / mL of influenza virus.

実施例３ 本発明の核酸抽出／増幅試薬に用いるシクロデキストリンの検討
以下の方法により、本発明の核酸抽出／増幅試薬に添加するシクロデキストリンの種類を検討した。
（１）実施例１で調製したＡ型（Ｈ１Ｎ１亜型）インフルエンザウイルス標準ＲＮＡ（配列番号１）を、注射用水を用いて１０^３コピー／５μＬとなるように希釈し、ＲＮＡ試料として用いた。
（２）以下の組成からなる反応液１５μＬを０．５ｍＬ容量ＰＣＲチューブ（Ｉｎｄｉｖｉｄｕａｌ Ｄｏｍｅ Ｃａｐ ＰＣＲ Ｔｕｂｅ、ＳＳＩ製）に分注した後、前記ＲＮＡ試料５μＬを添加した。

Example 3 Examination of cyclodextrin used in the nucleic acid extraction / amplification reagent of the present invention The type of cyclodextrin added to the nucleic acid extraction / amplification reagent of the present invention was examined by the following method.
(1) Form A prepared in Example 1 (H1N1 subtype) influenza virus standard RNA (SEQ ID NO: 1), water for injection and diluted to 10 ³ copies / 5 [mu] L was used used as the RNA sample.
(2) 15 μL of the reaction solution having the following composition was dispensed into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and then 5 μL of the RNA sample was added.

Composition of reaction solution: The concentration is the final concentration of 60 mM Tris-HCl (pH 8.6) after the addition of the initiator (in 30 μL).
0.25 mM dATP, dCTP, dGTP, dTTP, respectively
2.6 mM ATP, CTP, UTP, GTP respectively
3.06 mM ITP
70 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase 20 nM each INAF probe (prepared in Example 2) (SEQ ID NOs: 4 and 5)
First Primer at Concentrations Listed in Table 2 Second Primer at Concentrations Listed in Table 2 Cyclodextrin at the concentration listed in Table 4.

(3) The above reaction solution was kept warm at 46 ° C. for 4 minutes, and then 10 μL of an initiator having the following composition was added.

Initiator composition: The concentration is the final concentration after addition of the initiator (in 30 μL) 21.2 mM Magnesium chloride 102.7 mM Potassium chloride
1.2% glycerol 11.5% DMSO.

(4) Continue to use a fluorescence spectrophotometer with a temperature control function (TRCRapid-160, manufactured by Tosoh) that can directly measure the PCR tube, and react at 46 ° C. At the same time, the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm). ) Was measured over time for 30 minutes. A positive determination was made when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.2, with the addition of the starting solution as 0 minutes.

The results are shown in Table 4. It was confirmed that the reaction proceeded even when α-cyclodextrin (α-CD) or γ-cyclodextrin (γ-CD) was added to the nucleic acid amplification reagent. In particular, since γ-cyclodextrin has little effect on the nucleic acid amplification reaction even when added at a high concentration, γ-CD was used in the following experiments.

実施例４ 本発明の核酸抽出／増幅試薬に用いる界面活性剤の検討
以下の方法により、本発明の核酸抽出／増幅試薬に添加する界面活性剤の種類を検討した。
（１）実施例１で調製したＡ型（Ｈ１Ｎ１亜型）インフルエンザウイルス標準ＲＮＡを、注射用水を用いて１０^３コピー／２．５μＬとなるように希釈し、ＲＮＡ試料として用いた。
（２）表１に示すＡ型（Ｈ１Ｎ１亜型）インフルエンザウイルスの溶液（ＺｅｐｔｏＭｅｔｒｉｘ製）を注射用水で４．７×１０^０ＴＣＩＤ_５０／ｍＬに希釈することでウイルス試料を調製した。
（３）（１）で調製したＲＮＡ試料または（２）で調製したウイルス試料２．５μＬを、予め４６℃に加熱した以下の組成からなる界面活性剤を含む溶液（以下、ウイルス抽出液と表記）１２．５μＬに添加、撹拌し、４６℃で４分間保温することで、ウイルスＲＮＡ抽出物１５μＬを調製した。

Example 4 Examination of Surfactant Used in Nucleic Acid Extraction / Amplification Reagent of the Present Invention The type of surfactant to be added to the nucleic acid extraction / amplification reagent of the present invention was examined by the following method.
(1) Type A (H1N1 subtype) influenza virus standard RNA prepared in Example 1, diluted to 10 ³ copies /2.5μL with water for injection was used as an RNA sample.
(2) The virus samples were prepared by diluting the 4.7 × ¹⁰ 0 _TCID 50 / mL with water for injection solution (manufactured by Zeptometrix) of type A (H1N1 subtype) influenza virus shown in Table 1.
(3) A solution containing 2.5 μL of the RNA sample prepared in (1) or the virus sample prepared in (2) preheated to 46 ° C. and having the following composition (hereinafter referred to as virus extract). ) 15 μL of virus RNA extract was prepared by adding to 12.5 μL, stirring, and incubating at 46 ° C. for 4 minutes.

Composition of virus extract: Concentration is final concentration 42.4 mM magnesium chloride 205.4 mM potassium chloride 2.3% glycerol 23.0% DMSO after addition of virus sample (in 15 μL)
Surfactants at the concentrations listed in Table 5.

(4) Dispense 15 μL of a reaction solution containing γ-CD having the following composition into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), incubate at 46 ° C. for 4 minutes, and then in (3). 15 μL of the resulting viral RNA extract was immediately added. For the first primer and the second primer, a combination of oligonucleotides having the sequences and concentrations shown in Table 2 was used. Further, in the first primer, a T7 promoter sequence (SEQ ID NO: 6) is added to the 5'terminal side of the nucleotide sequence shown in each SEQ ID NO: shown in Table 2.

Reaction solution composition: Concentration is the final concentration 60 mM Tris-HCl (pH 8.6) after addition of viral RNA extract (in 30 μL).
0.25 mM dATP, dCTP, dGTP, dTTP, respectively
2.6 mM ATP, CTP, UTP, GTP respectively
3.06 mM ITP
70 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase 20 nM each INAF probe (prepared in Example 2) (SEQ ID NOs: 4 and 5)
First Primer at Concentrations Listed in Table 2 Second Primer 1 (w / w)% γ-CD at the concentration listed in Table 2.

(5) Continue to use a fluorescence spectrophotometer with a temperature control function (TRCRapid-160, manufactured by Tosoh) that can directly measure the PCR tube, and react at 46 ° C. At the same time, the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm). ) Was measured over time for 30 minutes. A positive determination was made when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.2, with the addition of the starting solution as 0 minutes.

The results when using the RNA sample and when using the virus sample are shown in Table 5. From the results when using RNA samples, it was confirmed that γ-cyclodextrin can alleviate the inhibition of the nucleic acid amplification reaction against various surfactants. In addition, based on the results of using a virus sample, nucleic acids can be extracted from the virus with high efficiency by using an anionic surfactant, especially a surfactant having a steroid skeleton such as sodium deoxycholate or sodium cholate. It was shown to be amplifyable.

実施例５ 本発明の核酸抽出試薬の保存安定性評価
実施例４で調製したウイルス抽出液を、室温で１２時間静置し、保存安定性を評価した。結果を表６に示す。ＳＤＳ、ＬＤＳ、デオキシコール酸ナトリウムを用いた場合には、結晶状の沈殿が形成された。一方、コール酸ナトリウム、タウロコール酸ナトリウム、タウロウルソデオキシコール酸ナトリウム、グリココール酸ナトリウムを用いた場合は、沈殿の形成は認められず、本発明における好ましい界面活性剤であることが分かった。

Example 5 Evaluation of storage stability of the nucleic acid extraction reagent of the present invention The virus extract prepared in Example 4 was allowed to stand at room temperature for 12 hours to evaluate the storage stability. The results are shown in Table 6. When SDS, LDS and sodium deoxycholate were used, a crystalline precipitate was formed. On the other hand, when sodium cholic acid, sodium taurocorate, sodium tauroursodeoxycholic acid, and sodium glycocholate were used, no precipitation was observed, and it was found to be a preferable surfactant in the present invention.

実施例６ 本発明の核酸抽出／増幅試薬に用いるシクロデキストリンの濃度の検討
以下の方法により、本発明の核酸抽出／増幅試薬に添加するシクロデキストリンの濃度を検討した。
（１）実施例１で調製したＡ型（Ｈ１Ｎ１亜型）インフルエンザウイルス標準ＲＮＡを、注射用水を用いて１０^３コピー／２．５μＬとなるように希釈し、ＲＮＡ試料として用いた。
（２）（１）で調製したＲＮＡ試料２．５μＬを、予め４６℃に加熱した以下の組成からなる界面活性剤を含む溶液（以下、ウイルス抽出液と表記）１２．５μＬに添加、撹拌し、４６℃で４分間保温することで、ウイルスＲＮＡ抽出物１５μＬを調製した。

Example 6 Examination of the concentration of cyclodextrin used in the nucleic acid extraction / amplification reagent of the present invention The concentration of cyclodextrin added to the nucleic acid extraction / amplification reagent of the present invention was examined by the following method.
(1) Type A (H1N1 subtype) influenza virus standard RNA prepared in Example 1, diluted to 10 ³ copies /2.5μL with water for injection was used as an RNA sample.
(2) Add 2.5 μL of the RNA sample prepared in (1) to 12.5 μL of a solution containing a surfactant having the following composition preheated to 46 ° C. (hereinafter referred to as virus extract) and stir. , 46 ° C. for 4 minutes to prepare 15 μL of viral RNA extract.

Composition of virus extract: Concentration is final concentration 42.4 mM magnesium chloride 205.4 mM potassium chloride 2.3% glycerol 23.0% DMSO after addition of virus sample (in 15 μL)
Surfactants at the concentrations listed in Tables 7-8.

(3) Dispense 15 μL of a reaction solution containing γ-CD having the following composition into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), incubate at 46 ° C. for 4 minutes, and then in (2). 15 μL of the resulting viral RNA extract was immediately added. For the first primer and the second primer, a combination of oligonucleotides having the sequences and concentrations shown in Table 2 was used. Further, in the first primer, a T7 promoter sequence (SEQ ID NO: 6) is added to the 5'terminal side of the nucleotide sequence shown in each SEQ ID NO: shown in Table 2.

Reaction solution composition: Concentration is the final concentration 60 mM Tris-HCl (pH 8.6) after addition of viral RNA extract (in 30 μL).
0.25 mM dATP, dCTP, dGTP, dTTP, respectively
2.6 mM ATP, CTP, UTP, GTP respectively
3.06 mM ITP
70 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase 20 nM each INAF probe (prepared in Example 2) (SEQ ID NOs: 4 and 5)
First Primer at Concentrations Listed in Table 2 Second Primer at Concentrations Listed in Table 2 γ-CD at concentrations listed in Tables 7-8.

(4) Continue to use a fluorescence spectrophotometer with a temperature control function (TRCRapid-160, manufactured by Tosoh) that can directly measure the PCR tube, and react at 46 ° C. At the same time, the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm). ) Was measured over time for 30 minutes. A positive determination was made when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.2, with the addition of the starting solution as 0 minutes.

Table 7 shows the results when sodium tauroursodeoxycholic acid was added as a surfactant, and Table 8 shows the results when sodium cholic acid was added. From the results in Tables 7 and 8, it can be seen that the inhibition of the nucleic acid amplification reaction can be sufficiently alleviated when cyclodextrin is contained at a concentration of 3 times or more, preferably 6 times or more the concentration of the surfactant having a steroid skeleton. Understand.

実施例７ 本発明の核酸抽出／増幅試薬に用いる界面活性剤の濃度の検討
以下の方法により、本発明の核酸抽出／増幅試薬に添加する界面活性剤の濃度を検討した。
（１）表１に示すＡ型（Ｈ１Ｎ１亜型）インフルエンザウイルスの溶液（ＺｅｐｔｏＭｅｔｒｉｘ製）を注射用水で４．７×１０^−１ＴＣＩＤ_５０／ｍＬに希釈することでウイル
ス試料を調製した。
（２）（１）で調製したウイルス試料２．５μＬを、予め４６℃に加熱した以下の組成からなる界面活性剤を含む溶液（以下、ウイルス抽出液と表記）１２．５μＬに添加、撹拌し、４６℃で４分間保温することで、ウイルスＲＮＡ抽出物１５μＬを調製した。

Example 7 Examination of the concentration of the surfactant used in the nucleic acid extraction / amplification reagent of the present invention The concentration of the surfactant added to the nucleic acid extraction / amplification reagent of the present invention was examined by the following method.
(1) A virus sample was prepared by diluting a solution of type A (H1N1 subtype) influenza virus (manufactured by ZeptoMetalix) shown in Table 1 with water for injection to 4.7 × 10 ^-1 TCID ₅₀ / mL.
(2) Add 2.5 μL of the virus sample prepared in (1) to 12.5 μL of a solution containing a surfactant having the following composition preheated to 46 ° C. (hereinafter referred to as virus extract) and stir. , 46 ° C. for 4 minutes to prepare 15 μL of viral RNA extract.

Composition of virus extract: Concentration is final concentration 42.4 mM magnesium chloride 205.4 mM potassium chloride 2.3% glycerol 23.0% DMSO after addition of virus sample (in 15 μL)
Sodium cholic acid at the concentrations listed in Table 9.

(3) Dispense 15 μL of a reaction solution containing γ-CD having the following composition into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), incubate at 46 ° C. for 4 minutes, and then in (2). 15 μL of the resulting viral RNA extract was immediately added. For the first primer and the second primer, a combination of oligonucleotides having the sequences and concentrations shown in Table 2 was used. Further, in the first primer, a T7 promoter sequence (SEQ ID NO: 6) is added to the 5'terminal side of the nucleotide sequence shown in each SEQ ID NO: shown in Table 2.

Reaction solution composition: Concentration is the final concentration 60 mM Tris-HCl (pH 8.6) after addition of viral RNA extract (in 30 μL).
0.25 mM dATP, dCTP, dGTP, dTTP, respectively
2.6 mM ATP, CTP, UTP, GTP respectively
3.06 mM ITP
70 mM trehalose 6 (w / w)% γ-CD
9.1U AMV Reverse Transcriptase 142U T7 RNA polymerase 20nM INAF probe each (prepared in Example 2) (SEQ ID NOs: 4 and 5)
First primer with the concentration shown in Table 2 Second primer with the concentration shown in Table 2.

(4) Continue to use a fluorescence spectrophotometer with a temperature control function (TRCRapid-160, manufactured by Tosoh) that can directly measure the PCR tube, and react at 46 ° C. At the same time, the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm). ) Was measured over time for 30 minutes. A positive determination was made when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.2, with the addition of the starting solution as 0 minutes.

The results are shown in Table 9. It can be seen that the viral nucleic acid can be satisfactorily extracted and amplified by using an extract containing sodium cholic acid, preferably an extract containing 0.8 (w / w)% or more of sodium cholic acid.

実施例８ 非イオン性界面活性剤をさらに含む核酸抽出試薬の検討
以下の方法により、ステロイド骨格を有する界面活性剤に加え、さらに非イオン性界面活性剤を添加した抽出試薬について検討を行った。
（１）表１に示すＡ型（Ｈ１Ｎ１亜型）インフルエンザウイルスの溶液（ＺｅｐｔｏＭｅｔｒｉｘ製）を注射用水で４．７×１０^−１ＴＣＩＤ_５０／ｍＬに希釈することでウイル
ス試料を調製した。
（２）（１）で調製したウイルス試料２．５μＬを、予め４６℃に加熱した以下の組成からなるコール酸ナトリウムおよびＴｗｅｅｎ ２０を含む溶液（以下、ウイルス抽出液と表記）１２．５μＬに添加、撹拌し、４６℃で４分間保温することで、ウイルスＲＮＡ抽出物１５μＬを調製した。

Example 8 Examination of Nucleic Acid Extraction Reagent Containing Nonionic Surfactant The extraction reagent to which the nonionic surfactant was further added in addition to the surfactant having a steroid skeleton was examined by the following method.
(1) A virus sample was prepared by diluting a solution of type A (H1N1 subtype) influenza virus (manufactured by ZeptoMetalix) shown in Table 1 with water for injection to 4.7 × 10 ^-1 TCID ₅₀ / mL.
(2) Add 2.5 μL of the virus sample prepared in (1) to 12.5 μL of a solution containing sodium cholic acid and Tween 20 having the following composition preheated to 46 ° C. (hereinafter referred to as virus extract). , Stirred and kept warm at 46 ° C. for 4 minutes to prepare 15 μL of viral RNA extract.

Composition of virus extract: Concentration is final concentration 42.4 mM magnesium chloride 205.4 mM potassium chloride 2.3% glycerol 23.0% DMSO after addition of virus sample (in 15 μL)
0.1 (v / v)% Tween 20
Surfactants at the concentrations shown in Table 10.

(3) Dispense 15 μL of a reaction solution containing γ-CD having the following composition into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), incubate at 46 ° C. for 4 minutes, and then in (2). 15 μL of the resulting viral RNA extract was immediately added. For the first primer and the second primer, a combination of oligonucleotides having the sequences and concentrations shown in Table 2 was used. Further, in the first primer, a T7 promoter sequence (SEQ ID NO: 6) is added to the 5'terminal side of the nucleotide sequence shown in each SEQ ID NO: shown in Table 2.

Reaction solution composition: Concentration is the final concentration 60 mM Tris-HCl (pH 8.6) after addition of viral RNA extract (in 30 μL).
0.25 mM dATP, dCTP, dGTP, dTTP, respectively
2.6 mM ATP, CTP, UTP, GTP respectively
3.06 mM ITP
70 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase 20 nM each INAF probe (prepared in Example 2) (SEQ ID NOs: 4 and 5)
First Primer at Concentrations Listed in Table 2 Second Primer at Concentrations Listed in Table 2 γ-CD at the concentration listed in Table 10.

(5) Continue to use a fluorescence spectrophotometer with a temperature control function (TRCRapid-160, manufactured by Tosoh) that can directly measure the PCR tube, and react at 46 ° C. At the same time, the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm). ) Was measured over time for 30 minutes. A positive determination was made when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.2, with the addition of the starting solution as 0 minutes. The results are shown in Table 10.

It can be seen that the viral nucleic acid can be successfully extracted and amplified in the range of the concentration of sodium cholic acid in the extract in the range of 0.4 to 1.6 (w / w)%. Further, it can be seen that the nucleic acid extraction / amplification efficiency is improved as compared with the extraction reagent (Table 9) to which Tween 20 is not added.

実施例９ 本発明の核酸抽出試薬による抽出時間の検出
以下の方法により、本発明の核酸抽出／増幅試薬を用いた場合の抽出時間について、検討を行った。

Example 9 Detection of Extraction Time with Nucleic Acid Extraction Reagent of the Present Invention The extraction time when the nucleic acid extraction / amplification reagent of the present invention was used was examined by the following method.

(1) Table A-type shown in 1 (H1N1 subtype) of influenza virus solution (manufactured by Zeptometrix), the virus samples were prepared by diluting the 4.7 × ¹⁰ 0 _TCID 50 / mL in water for injection.
(2) Add 2.5 μL of the virus sample prepared in (1) to 12.5 μL of a solution containing a surfactant having the following composition preheated to 46 ° C. (hereinafter referred to as virus extract) and stir. , 46 ° C. for 0-4 minutes to prepare 15 μL of viral RNA extract.

Composition of virus extract: Concentration is final concentration 42.4 mM magnesium chloride 205.4 mM potassium chloride 2.3% glycerol 23.0% DMSO after addition of virus sample (in 15 μL)
0.1 (v / v)% Tween 20
1.0 (w / w)% sodium cholic acid.

(3) Dispense 15 μL of a reaction solution containing γ-CD having the following composition into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), incubate at 46 ° C. for 4 minutes, and then in (2). 15 μL of the resulting viral RNA extract was immediately added. For the first primer and the second primer, a combination of oligonucleotides having the sequences and concentrations shown in Table 2 was used. Further, in the first primer, a T7 promoter sequence (SEQ ID NO: 6) is added to the 5'terminal side of the nucleotide sequence shown in each SEQ ID NO: shown in Table 2.

Reaction solution composition: Concentration is the final concentration 60 mM Tris-HCl (pH 8.6) after addition of viral RNA extract (in 30 μL).
0.25 mM dATP, dCTP, dGTP, dTTP, respectively
2.6 mM ATP, CTP, UTP, GTP respectively
3.06 mM ITP
70 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase 20 nM each INAF probe (prepared in Example 2) (SEQ ID NOs: 4 and 5)
First Primer at Concentrations Listed in Table 2 Second Primer 6 (w / w)% γ-CD at the concentration listed in Table 2.

(5) Continue to use a fluorescence spectrophotometer with a temperature control function (TRCRapid-160, manufactured by Tosoh) that can directly measure the PCR tube, and react at 46 ° C. At the same time, the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm). ) Was measured over time for 30 minutes. A positive determination was made when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.2, with the addition of the starting solution as 0 minutes.

The results are shown in Table 11. Even when the heating time at the time of extraction was set to 0 minutes (a method in which the virus was mixed with the extraction reagent and then immediately mixed with the reaction solution), the virus nucleic acid could be successfully extracted / amplified.

実施例１０ 本発明の核酸抽出／増幅試薬によるインフルエンザウイルスの検出感度
以下の方法により、本発明の核酸抽出／増幅試薬を用いて、各種インフルエンザウイルスの検出を行った。
（１）表１に示すＡ型（Ｈ１Ｎ１亜型）インフルエンザウイルス、Ａ型（Ｈ３Ｎ２亜型）インフルエンザウイルス、Ｂ型インフルエンザウイルスの溶液（ＺｅｐｔｏＭｅｔｒｉｘ製）を、注射用水で表１２に記載の濃度に希釈することでウイルス試料を調製した。
（２）（１）で調製したウイルス試料２．５μＬを、予め４６℃に加熱した以下の組成からなる界面活性剤を含む溶液（以下、ウイルス抽出液と表記）１２．５μＬに添加、撹拌し、４６℃で４分間保温することで、ウイルスＲＮＡ抽出物１５μＬを調製した。

Example 10 Sensitivity of Influenza Virus Detection by Nucleic Acid Extraction / Amplification Reagent of the Present Invention Various influenza viruses were detected using the nucleic acid extraction / amplification reagent of the present invention by the following method.
(1) A solution of influenza A (H1N1 subtype) virus, type A (H3N2 subtype) influenza virus, and type B influenza virus (manufactured by ZeptoMetalix) shown in Table 1 is diluted with water for injection to the concentration shown in Table 12. A virus sample was prepared by the above.
(2) Add 2.5 μL of the virus sample prepared in (1) to 12.5 μL of a solution containing a surfactant having the following composition preheated to 46 ° C. (hereinafter referred to as virus extract) and stir. , 46 ° C. for 4 minutes to prepare 15 μL of viral RNA extract.

Composition of virus extract: Concentration is final concentration 42.4 mM magnesium chloride 205.4 mM potassium chloride 2.3% glycerol 23.0% DMSO after addition of virus sample (in 15 μL)
0.1 (v / v)% Tween 20
1.0 (w / w)% sodium cholic acid.

(3) Dispense 15 μL of a reaction solution containing γ-CD having the following composition into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), incubate at 46 ° C. for 4 minutes, and then in (2). 15 μL of the resulting viral RNA extract was immediately added. For the first primer and the second primer, a combination of oligonucleotides having the sequences and concentrations shown in Table 2 was used. Further, in the first primer, a T7 promoter sequence (SEQ ID NO: 6) is added to the 5'terminal side of the nucleotide sequence shown in each SEQ ID NO: shown in Table 2.

Reaction solution composition: Concentration is the final concentration 60 mM Tris-HCl (pH 8.6) after addition of viral RNA extract (in 30 μL).
0.25 mM dATP, dCTP, dGTP, dTTP, respectively
2.6 mM ATP, CTP, UTP, GTP respectively
3.06 mM ITP
70 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase 20 nM each INAF probe (prepared in Example 2) (SEQ ID NOs: 4 and 5)
First Primer at Concentrations Listed in Table 2 Second Primer 6 (w / w)% γ-CD at the concentration listed in Table 2.

(5) Continue to use a fluorescence spectrophotometer with a temperature control function (TRCRapid-160, manufactured by Tosoh) that can directly measure the PCR tube, and react at 46 ° C. At the same time, the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm). ) Was measured over time for 30 minutes. A positive determination was made when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.2, with the addition of the starting solution as 0 minutes.

The results are shown in Table 12. Generally are able to detect 4.7 × ¹⁰ -2 _TCID 50 / mL of influenza virus, yet simple and fast way, the same high efficiency nucleic acid extraction / purification methods using spin columns (Reference Example 1) Was found to have.

実施例１１ 凍結乾燥増幅試薬の調整方法
以下の組成からなるγ−ＣＤを含む試薬液１８．５μＬを０．５ｍＬ容量ＰＣＲチューブ（Ｉｎｄｉｖｉｄｕａｌ Ｄｏｍｅ Ｃａｐ ＰＣＲ Ｔｕｂｅ、ＳＳＩ製）に分注し、Ｖｉｒｔｉｓ ＧＥＮＥＳＩＳ １２ＳＬにて、−４０℃ ２時間、−２０℃ １４時間、２５℃ ２時間で真空下において凍結乾燥した。なお、第一のプライマーおよび第二のプライマーには、表２に示す配列および濃度からなるオリゴヌクレオチドの組み合わせを用いた。また第一のプライマーには、表２に示した各配列番号に記載の塩基配列の５’末端側に、Ｔ７プロモーター配列（配列番号６）が付加されている。

Example 11 Method for preparing freeze-dried amplification reagent 18.5 μL of a reagent solution containing γ-CD having the following composition was dispensed into a 0.5 mL volume PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and Virtis GENESIS 12SL. Freeze-dried under vacuum at −40 ° C. for 2 hours, −20 ° C. for 14 hours, and 25 ° C. for 2 hours. For the first primer and the second primer, a combination of oligonucleotides having the sequences and concentrations shown in Table 2 was used. Further, in the first primer, a T7 promoter sequence (SEQ ID NO: 6) is added to the 5'end side of the nucleotide sequence shown in each SEQ ID NO: shown in Table 2.

Composition of reagent solution: Concentration is the final concentration 60 mM Tris-HCl (pH 8.6) after addition of viral RNA extract (in 30 μL).
0.25 mM dATP, dCTP, dGTP, dTTP, respectively
2.7 mM ATP, CTP, UTP, GTP respectively
3.06 mM ITP
198.9 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase 20 nM each INAF probe (prepared in Example 2) (SEQ ID NOs: 4 and 5)
First Primer at Concentrations listed in Table 2 Second Primer at concentrations listed in Table 2 6.8 (w / w)% γ-CD.

Example 12 Influenza virus detection sensitivity by freeze-dry amplification reagent Various influenza viruses were detected using the nucleic acid extraction / amplification reagent of the present invention by the following method.
(1) A virus sample was prepared by diluting the type A (H1N1 subtype) influenza virus shown in Table 1 with water for injection to the concentration shown in Table 13.
(2) Add 3 μL of the virus sample prepared in (1) to 27 μL of a solution containing a surfactant having the following composition preheated to 46 ° C. (hereinafter referred to as virus extract), stir, and at 46 ° C. After keeping warm for 4 minutes, 30 μL was added to the freeze-drying reagent prepared in Example 11.

Composition of virus extract: Concentration is final concentration after addition of virus sample 22.2 mM Magnesium chloride 47.5 mM Potassium chloride 1.2% Glycerol 10.3% DMSO
0.05 (v / v)% Tween 20
1.5 (w / w)% sodium cholic acid.

At the same time, using the liquid reagent (reaction solution) shown in Example 10, a fluorescence spectrophotometer with a temperature control function (TRCRapid-160, manufactured by Tosoh) was used to react at 46 ° C., and at the same time, the fluorescence intensity (excitation) of the reaction solution was excited. Wavelength 470 nm, fluorescence wavelength 520 nm) was measured over time for 30 minutes. A positive determination was made when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.2, with the addition of the starting solution as 0 minutes.

The results are shown in Table 13. It was shown that the amplification reagent in the dry state also has the same detection performance as the liquid reagent.

Claims (10)

Contain the following (i) (ii) (iii ), a kit for extracting and amplifying the viral nucleic acid from a sample containing the virus,
The cyclodextrin is γ-cyclodextrin,
The surfactant is either cholic acid, taurocholic acid, glycocholic acid, tauroursodeoxycholic acid or a salt thereof.
A kit in which the concentration of cyclodextrin (w / w)% is 3 times or more the concentration of a surfactant having a steroid skeleton (w / w)% .
(I) Viral nucleic acid extraction reagent containing at least a surfactant having a steroid skeleton (ii) Cyclodextrin (iii) nucleic acid amplification reagent The kit for extracting and amplifying the viral nucleic acid according to claim 1, wherein the cyclodextrin is contained in a nucleic acid amplification reagent. The kit for extracting and amplifying the viral nucleic acid according to claim 1 or 2, wherein the virus is an enveloped virus. A kit for extracting and amplifying the viral nucleic acid according to any one of claims 1 to 3, wherein the virus is an influenza virus. The kit for extracting and amplifying the viral nucleic acid according to any one of claims 1 to 4 , wherein the concentration of the surfactant having a steroid skeleton in the extraction reagent is 0.4 (w / w)% or more. A kit for extracting and amplifying the viral nucleic acid according to any one of claims 1 to 5 , further comprising a nonionic surfactant. A kit for extracting and amplifying the viral nucleic acid according to any one of claims 1 to 6, wherein the nucleic acid amplification reagent is in a dry form. Includes the following of (i) (ii) (iii ) step, a method of extracting and amplifying the viral nucleic acid from a sample containing the virus,
The cyclodextrin is γ-cyclodextrin,
The surfactant is either cholic acid, taurocholic acid, glycocholic acid, tauroursodeoxycholic acid or a salt thereof.
A method in which the concentration (w / w)% of cyclodextrin is at least 3 times the concentration (w / w)% of a surfactant having a steroid skeleton .
(I) the virus, contacting the cyclodextrin viral nucleic acid extract extracted by a nucleic acid extraction step of contacting at least containing viral nucleic acid extraction reagent a surfactant having a steroid skeleton (ii) (i) (iii ) Nucleic acid amplification step of contacting a part or all of the solution obtained in the step (ii) with a nucleic acid amplification reagent. Includes the following of (i) (ii) step, a method of extracting and amplifying the viral nucleic acid from a sample containing the virus,
The cyclodextrin is γ-cyclodextrin,
The surfactant is either cholic acid, taurocholic acid, glycocholic acid, tauroursodeoxycholic acid or a salt thereof.
A method in which the concentration (w / w)% of cyclodextrin is at least 3 times the concentration (w / w)% of a surfactant having a steroid skeleton .
(I) the virus, the nucleic acid extraction step (ii) viral nucleic acid extract extracted by (i) contacting a viral nucleic acid extraction reagent containing at least a surfactant having a steroid skeleton, a nucleic acid amplification reagent comprising a cyclodextrin Nucleic acid amplification step to contact with The method of claim 8 or 9, wherein the virus is an influenza virus.