JP2018000124A - Kit for nucleic acid extraction and amplification from virus and extraction and amplification method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a viral nucleic acid extraction/amplification reagents characterized by extracting nucleic acid from a virus simply, rapidly, and highly efficiently, and not inhibiting nucleic acid amplification reaction; and to provide nucleic acid extraction or amplification methods using the reagent.SOLUTION: In a viral nucleic acid extraction and amplification kit having i) a viral nucleic acid extracting reagent, and ii) a viral nucleic acid amplification reagent, above mentioned i) a viral nucleic acid extracting reagent or ii) a viral nucleic acid amplification reagent contains a reducing agent, a polysaccharide degrading enzyme, and a dummy nucleic acid, and extraction and amplification are conducted using the kit for amplifying without purification after extraction of viral nucleic acid.SELECTED DRAWING: None

Description

  The present invention relates to a kit for extracting and amplifying a nucleic acid from a virus in a simple, rapid and highly efficient manner, and a nucleic acid extraction and amplification method using the kit.

  In the field of clinical examination of infectious diseases, it is important to detect pathogens simply, quickly and with high sensitivity for the purpose of obtaining accurate test results during outpatient treatment and providing appropriate treatment. For example, in the inspection of influenza virus infections, which are prevalent every winter, rapid therapeutic kits are widely used because there may be therapeutic agents that exert high effects when administered at the early stage of infection. As the principle of such a rapid inspection method, a detection method by an 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. 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 is known. Since the detection method using the nucleic acid amplification method specifically detects and detects the nucleic acid contained in the pathogen, the pathogen can be detected quickly and with high sensitivity.

  When a pathogen is detected by a detection method using a nucleic acid amplification method, an operation of extracting nucleic acid from a sample containing the pathogen is generally performed in advance. Known methods for extracting nucleic acids from pathogens, particularly viruses, include methods using protein denaturing agents such as surfactants and guanidine, and organic solvents such as phenol and chloroform (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 necessary after the extraction step, which requires complicated and time-consuming operations. In order to simplify such nucleic acid extraction and purification operations, various kits and devices are commercially available. For example, a kit that uses a reagent such as EXTRAGEN II (manufactured by Tosoh) and a centrifugal separator, a kit that uses a spin column such as QIAamp Virtual RNA Mini Kit (manufactured by Qiagen), and a magnet such as a chemical prepit (manufactured by PerkinElmer) Examples thereof include an automatic extraction device using beads. However, even if the above-described kit or device is used, it takes about 10 minutes to 1 hour, requires a lot of work, and requires special devices such as a centrifuge and an automatic extraction device. A method was sought.

  On the other hand, a method for detecting a target nucleic acid by performing nucleic acid amplification without extracting and purifying the nucleic acid as in the method described in Non-Patent Document 2 is also known, but the sensitivity is insufficient. It was. As a method for performing nucleic acid extraction operation and subjecting it to a nucleic acid amplification reaction without purification, there is a method in which a nucleic acid is extracted using a surfactant and then neutralized with cyclodextrin and used for the nucleic acid amplification step. Are known. In Patent Document 7, nucleic acid is extracted from yeast and Escherichia coli using 0.5% sodium dodecyl sulfate (SDS), and nucleic acid amplification is performed by PCR or RCA 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 simplicity, quickness, safety, and the like. The patent document also shows that yeast-derived genomic DNA can be amplified with a PCR reaction solution containing cyclodextrin even under conditions where a surfactant other than SDS is added. However, except for the case where SDS is used, no example of extracting nucleic acids from cells or viruses with a surfactant is shown.

  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, description about detection sensitivity and extraction efficiency is not recognized, and a reagent for extracting and amplifying nucleic acid from a virus with high efficiency is not known. Further, the nucleic acid amplification methods of Patent Documents 7 and 8 are methods for amplifying DNA at a relatively high temperature using a DNA-dependent DNA polymerase, and a nucleic acid amplification by a concerted reaction of RNA polymerase and reverse transcriptase at a relatively low temperature. The conditions for nucleic acid extraction and amplification from viruses in methods (TMA method, TRC method, NASBA method, etc.) that perform the above-mentioned were not known.

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

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

  It is an object of the present invention to provide a nucleic acid extraction and amplification kit that can be used for amplification without extraction and purification without difficulty and nucleic acid amplification, and that does not easily inhibit nucleic acid amplification reaction. is there.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have made contact with a reagent containing a reducing agent, a virus, and a specimen, thereby achieving high efficiency without performing complicated extraction and / or purification operations. The inventors have found that nucleic acid can be extracted and amplified in a specimen, and the present invention has been completed.

That is, the present invention includes the following embodiments:
[1]
In a viral nucleic acid extraction and amplification kit having i) a viral nucleic acid extraction reagent, and ii) a viral nucleic acid amplification reagent,
containing a reducing agent in i) viral nucleic acid extraction reagent or ii) viral nucleic acid amplification reagent,
And the kit for amplifying a viral nucleic acid without purification after extraction.
[2]
The kit according to [1] above, wherein i) viral nucleic acid extraction reagent or ii) viral nucleic acid amplification reagent contains a polysaccharide degrading enzyme and / or a dummy nucleic acid.
[3]
The kit according to [1] or [2] above, wherein the virus is an influenza virus.
[4]
The kit according to any one of [1] to [3] above, wherein the reducing agent is dithiothreitol or tris (2carboxyethyl) phosphine.
[5]
A sample containing a virus is brought into contact with i) a virus nucleic acid extraction reagent and ii) a virus nucleic acid amplification reagent of the kit according to any one of claims 1 to 4, and the virus nucleic acid is amplified without being purified after extraction. A method for extracting and amplifying viral nucleic acid.

  Hereinafter, the present invention will be described in detail.

  In the present invention, the reducing agent has a reducing action. As an example, dithiothreitol (hereinafter referred to as DTT), tris (2-carboxyethyl) phosphine (hereinafter referred to as TCEP), 2-mercaptoethanol, tributylphosphine, iodoacetamide, iron (II), N-acetyl-L-cysteine ( Hereafter, NALC) etc. can be illustrated. In addition, although it does not show a reducing action by nature, those that show a reducing action in the reagent are also included. The reducing agent is preferably NALC, DTT or TCEP, more preferably DTT or TCEP. The concentration is not particularly limited, but the upper limit is up to a concentration that inhibits the nucleic acid amplification reaction. Moreover, it is preferable to add a pH adjuster (buffer solution or the like) because the upper limit is increased.

  In the present invention, the polysaccharide degrading enzyme is an enzyme having a function of degrading polysaccharide as a result. Examples include β-galactosidase, β-glucuronidase, α-L-iduronidase, iduronic acid-2-sulfatase, iduronic acid-2-sulfatase, acetyl-CoA, α-glucosaminide N-acetyltransferase, sulfamidase, galactose-6 -Sulphatase, N-acetylgalactosamine-4-sulfatase, hyaluronidase and the like are included, and semi-alkaline phosphatase can also be exemplified.

  In the present invention, the dummy nucleic acid is a nucleic acid other than the target viral nucleic acid, and mitigates the influence on the target nucleic acid amplification reaction by binding a substance that inhibits the nucleic acid amplification reaction of the target virus. The purpose is to do. Examples include tRNA, a mixture of 16S and 23S, PolyA and the like.

  The extraction target of the present invention may be any kind of viral nucleic acid. Viruses have a structure in which nucleic acids are included by a protein shell called capsid, and are clearly distinguished from bacteria, microorganisms, cells, and the like in terms of structure and size. Examples of preferable extraction targets in the present invention include viruses having an envelope and viruses having a single-stranded RNA. An enveloped virus refers to a virus whose viral capsid is covered by an envelope mainly composed of lipids, and examples include herpes simplex virus, influenza virus, RS virus, and AIDS virus (HIV). Viruses having single-stranded RNA belong to the fourth group (single-stranded RNA + strand), the fifth group (single-stranded RNA-strand), and the sixth group (single-stranded RNA reverse transcription) in the Baltimore classification. Examples thereof include coronavirus, RS virus, human metapneumovirus, influenza virus, and AIDS virus (HIV). Viruses having single-stranded RNA are preferred because they are suitable for nucleic acid amplification methods using RNA as a starting material, such as the TRC method. Among them, influenza virus is particularly preferable as a virus to be extracted in the extraction reagent of the present invention.

  In the present invention, a virus-containing sample includes a virus simply dissolved in water, physiological saline or a buffer, a virus-containing biological sample itself, a filter paper containing the biological sample, a swab, and the like. Examples of the biological sample include blood, stool, urine, sputum, lymph, plasma, ejaculate, lung aspirate, cerebrospinal fluid, pharyngeal wipe, nasal wipe, gargle, saliva, and tears.

  In the present invention, the reducing agent, polysaccharide degrading enzyme, and dummy nucleic acid may be contained in either i) viral nucleic acid extraction reagent or ii) viral nucleic acid amplification reagent, but i) viral nucleic acid extraction reagent. preferable.

  The viral nucleic acid extraction reagent of the present invention may further contain various components. Containing components necessary for subsequent 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, etc. This 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 acid. Examples of the organic solvent further contained in the extraction reagent of the present invention include dimethyl sulfoxide (DMSO). In addition, a surfactant, a protein denaturant, and an RNase inhibitor can be added as long as they do not interfere with the nucleic acid amplification reaction. Examples of surfactants to be added include surfactants having a steroid skeleton, anionic surfactants such as SDS, amphoteric surfactants such as CHAPS, Span 20 (trade name), MEGA-8 (product) Name), polyoxyethylene sorbitan fatty acid ester (Tween 20 (trade name), Tween 40 (trade name), Tween 60 (trade name), Tween 80 (trade name), etc.), etc.) . Among these, nonionic surfactants are preferable in that they have an effect of promoting the extraction of viral nucleic acids and have little influence on the nucleic acid amplification reaction. Tween 20 (trade name), which is a polyoxyethylene sorbitan fatty acid ester surfactant, is more preferred.

  In the present invention, a sample containing a virus is brought into contact with i) a virus nucleic acid extraction reagent and ii) a virus nucleic acid amplification reagent, but the order is not particularly limited, and the sample may be simultaneously brought into contact. Preferably, a sample containing virus is brought into contact with i) a viral nucleic acid extraction reagent and then contacted with ii) a viral nucleic acid amplification reagent. In that case, the contact time between the sample and i) the virus nucleic acid extraction reagent 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. When the agent is included in the i) virus nucleic acid extraction reagent, the virus nucleic acid can be sufficiently extracted within 4 minutes after the contact, and can be subjected to a subsequent step such as a step of contacting with the cyclodextrin immediately after the contact.

  In the present invention, the viral nucleic acid extract is obtained by bringing a virus-containing sample into contact with a viral nucleic acid extraction reagent.

  As an example of a preferred embodiment of the present invention, a viral nucleic acid is extracted from a specimen using a viral nucleic acid extraction reagent containing at least a reducing agent, a polysaccharide degrading enzyme, and a dummy nucleic acid, and converted into a nucleic acid amplification reagent without purifying the viral nucleic acid extract. A reagent for performing a nucleic acid amplification reaction when added can be mentioned. The concentration of cyclodextrin is not particularly limited.

  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 isothermally is preferable in that a relatively complicated apparatus for raising and lowering the temperature is unnecessary. Such a nucleic acid amplification method includes a first primer having a sequence homologous to part of a viral nucleic acid, a second primer having a sequence complementary to a part of the viral nucleic acid, and RNA-dependent DNA polymerase activity. A method for amplifying a target nucleic acid 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 ′ end side thereof (for example, TMA method, TRC method, NASBA Method). This method is a method of amplifying a nucleic acid using a single-stranded RNA as a target, but a double-stranded DNA or the like can also be amplified by using a method disclosed in JP-A-2015-11636.

  In the present invention, the above-mentioned i) viral nucleic acid extraction reagent and ii) viral nucleic acid amplification reagent are used as a kit, and the viral nucleic acid is amplified without being purified after extraction.

  According to the present invention, amplification can be performed without performing a purification operation after viral nucleic acid extraction, so that it can be carried out simply, rapidly and with high efficiency. Specifically, with the kit and method of the present invention, a complicated extraction step is unnecessary, and a detection sensitivity equivalent to that of QIAamp Viral RNA Mini can be achieved.

  Hereinafter, the present invention will be described in detail by examples when influenza virus is used as a virus, but the present invention is not limited by these examples.

Reference Example 1 Preparation of Standard RNA Influenza virus RNA (hereinafter referred to as standard RNA) used in Examples described later was prepared by the method shown below.
(1) SEQ ID NO: 1 (type A (H1N1 subtype) influenza virus, segment 5, cDNA partial sequence, GenBank No. FJ969536, positions 410 to 930), SEQ ID NO: 2 (type A (H3N2 subtype) influenza virus , Segment 5, cDNA partial sequence, 455th to 975th of GenBank No. NC — 007369 (where 663rd C is T)) and SEQ ID NO: 3 (influenza B virus, segment 7, cDNA partial sequence, GenBank No. CY115184 Double-stranded DNAs having the base sequences described in (No. 206 to 735) are inserted into T-Vector pMD20 (manufactured by Takara Bio Inc.) (note that the 5 ′ end side of the complementary strand of the cDNA sequence) SP6 Promo It is added ter).

(2) The plasmid DNA prepared in (1) was digested with restriction enzymes at the 5 ′ end side of the inserted influenza virus cDNA to prepare linear DNA.
(3) In vitro transcription with SP6 RNA polymerase was performed using the DNA prepared in (2) as a template. Thereafter, 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 full length of the standard RNA prepared in this example is about 500 bases, which is a part of the full length of influenza virus RNA (segment 5: about 1500 bases, segment 7: about 1100 bases). Is fully applicable.

参考例２ インターカレーター性蛍光色素標識核酸プローブの作製
Ｉｓｈｉｇｕｒｏらの方法（Ｉｓｈｉｇｕｒｏ，Ｔ．ｅｔ ａｌ，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．，２４，４９９２−４９９７（１９９６））により、配列番号４に記載の配列（ＧｅｎＢａｎｋ Ｎｏ．ＫＪ７４１９８９の７２４番目から７４６番目までの塩基配列の相補配列）の５’末端から１７番目のＴと１８番目のＴの間、および配列番号５に記載の配列（ＧｅｎＢａｎｋ Ｎｏ．ＣＹ１１５１８４の４６３番目から４８３番目までの塩基配列の相補配列）の５’末端から８番目のＡと９番目のＴの間のリン酸ジエステル部分にリンカーを介してチアゾールオレンジもしくはＢＯを結合させ、インターカレーター性蛍光色素で標識された核酸プローブ（以下、ＩＮＡＦプローブと表記）を調製した。構造式を化１に示す。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。

Reference Example 2 Preparation of Intercalator Fluorescent Dye-Labeled Nucleic Acid Probe The sequence described in SEQ ID NO: 4 (GenBank) by the method of Ishiguro et al. (Ishiguro, T. et al, Nucleic Acids Res., 24, 4992-4997 (1996)). No. KJ741989, the complementary sequence of the nucleotide sequence from the 724th to the 746th base sequence) between the 17th T and the 18th T from the 5 ′ end, and the sequence described in SEQ ID NO: 5 (GenBank No. CY115184, 463rd) Intercalating fluorescent dye by binding thiazole orange or BO to the phosphodiester part between the 8th A and 9th T from the 5 'end of the 5' to the 483th base sequence through a linker. A nucleic acid probe labeled with Over Breakfast and notation) was prepared. The structural formula is shown in Chemical Formula 1. ^{Wherein, B 1, B 2, B} 3, B 4 represents a base. In addition, in order to prevent the elongation reaction from 3 ′ terminal side —OH, 3 ′ terminal side —OH is modified with glycolic acid.

参考例３ 本発明に使用した臨床検体の入手
臨床検体は、各種医療機関により得た。インフルエンザウイルスに感染した疑いのある患者から、スワブにより咽頭や、鼻腔を擦過した擦過物、鼻かみ液、うがい液、鼻かみ液を採取し、イムノクロマト法によりＡ型、Ｂ型、又は陰性と判断した。

Reference Example 3 Obtaining Clinical Samples Used in the Present Invention Clinical samples were obtained from various medical institutions. From the patient suspected of being infected with influenza virus, swabs, nasal cavity, nasal discharge, gargle, and nasal discharge were collected by swab, and determined to be A, B, or negative by immunochromatography. did.

Comparative Example 1 Inhibition of Nucleic Acid Amplification Reaction by Clinical Specimen (1) The swab to which the nasal scrape obtained in Reference Example 3 was attached was suspended in 136 μL of water for injection. The suspension was used as a clinical specimen. The immunochromatography method was type A.
(2) 15 μL of a reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).
Composition of reaction solution: Concentration is 60 mM Tris-HCl buffer (pH 8.65) at final concentration after addition of extract (in 30 μL)
0.25 mM dATP, dCTP, dGTP, dTTP each
2.7 mM each ATP, CTP, UTP, GTP
3.06 mM ITP
60 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase The concentration of the INAF probe described in Table 3 (prepared in Reference Example 2) (SEQ ID NOs: 4 and 5)
6.8% cyclodextrin first primer at the concentration described in Table 3 second primer at the concentration described in Table 3

（３）上記の反応液を４６℃で４分間保温後、以下の組成からなる抽出液１５μＬを添加した。抽出液には、あらかじめ前記臨床検体を０μＬ、０．５μＬ、１．７μＬ、３μＬ、５μＬ、８μＬをそれぞれ添加した。
抽出液の組成：濃度は抽出液添加後（３０μＬ中）の最終濃度
２２．２ｍＭ 塩化マグネシウム
３５ｍＭ 塩化カリウム
１．２％ グリセロール
１０．３％ ＤＭＳＯ
０．０５％ Ｔｗｅｅｎ２０
１．５％ コール酸ナトリウム水溶液。

(3) After incubating the above reaction solution at 46 ° C. for 4 minutes, 15 μL of an extract solution having the following composition was added. To the extract, 0 μL, 0.5 μL, 1.7 μL, 3 μL, 5 μL, and 8 μL of the clinical specimen were previously added, respectively.
Composition of extract: Concentration is 22.2 mM magnesium chloride 35 mM potassium chloride after addition of extract (in 30 μL)
1.2% Glycerol 10.3% DMSO
0.05% Tween20
1.5% aqueous sodium cholate solution.

  (4) Subsequently, using a fluorescence spectrophotometer with a temperature control function (TRCReady-80, manufactured by Tosoh Corporation) capable of directly measuring a PCR tube, the reaction mixture was reacted at 46 ° C. and simultaneously the fluorescence intensity of the reaction solution was measured over time for 30 minutes. . The time when the extract was added was 0 minute, and the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value of the predetermined time by the fluorescence intensity ratio of the background) exceeded 1.2 was determined as positive, and the detection time and did.

  The results are shown in Table 4. It can be seen that increasing the amount of clinical specimen significantly inhibits the TRC reaction.

実施例１ 本発明の核酸抽出／増幅試薬に用いる添加剤の上限濃度１
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添加剤の濃度を検討した。
（１）参考例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に相当するもの）を、注射用水を用いて１０^４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。
反応液の組成：濃度は抽出液添加後（３０μＬ中）の最終濃度
６０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ８．６５）
各０．２５ｍＭ ｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰ、ｄＴＴＰ
各２．７ｍＭ ＡＴＰ、ＣＴＰ、ＵＴＰ、ＧＴＰ
３．０６ｍＭ ＩＴＰ
６０ｍＭ トレハロース
９．１Ｕ ＡＭＶ逆転写酵素
１４２Ｕ Ｔ７ ＲＮＡポリメラーゼ
表３に記載の濃度のＩＮＡＦプローブ（参考例２で調製）（配列番号４および５）
６．８％ シクロデキストリン
表３に記載の濃度の第一のプライマー
表３に記載の濃度の第二のプライマー。

Example 1 Upper limit concentration of additive used for nucleic acid extraction / amplification reagent of the present invention 1
The concentration of the additive added to the nucleic acid extraction / amplification reagent of the present invention was examined by the following method.
(1) A type A (H1N1 subtype) influenza virus standard RNA (corresponding to SEQ ID NO: 1) prepared in Reference Example 1 is diluted with water for injection to 10 ⁴ copies / 5 μL, and an RNA sample Used as.
(2) 15 μL of a reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).
Composition of reaction solution: Concentration is 60 mM Tris-HCl buffer (pH 8.65) at final concentration after addition of extract (in 30 μL)
0.25 mM dATP, dCTP, dGTP, dTTP each
2.7 mM each ATP, CTP, UTP, GTP
3.06 mM ITP
60 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase The concentration of the INAF probe described in Table 3 (prepared in Reference Example 2) (SEQ ID NOs: 4 and 5)
6.8% cyclodextrin First primer at the concentration described in Table 3 Second primer at the concentration described in Table 3.

(3) After incubating the above reaction solution at 46 ° C. for 4 minutes, 15 μL of an extract solution having the following composition was added. To the extract, 1.5 μL of the RNA sample was added in advance.
Composition of extract: Concentration is 22.2 mM magnesium chloride 35 mM potassium chloride after addition of extract (in 30 μL)
1.2% Glycerol 10.3% DMSO
0.05% Tween20
1.5% Sodium cholate Additives at various concentrations as listed in Table 5.

  (4) Using a fluorescence spectrophotometer with a temperature control function (TRCRapid-160, manufactured by Tosoh Corp.) capable of directly measuring a PCR tube, the reaction solution is reacted at 46 ° C. and simultaneously the fluorescence intensity of the reaction solution (excitation wavelength: 470 nm, fluorescence wavelength: 520 nm). ) Was measured over time for 30 minutes. The time when the extract was added was 0 minute, and when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value for a predetermined time by the fluorescence intensity ratio of the background) exceeded 1.2, the positive determination was made and the detection time was designated .

  The results are shown in Table 5.

実施例２ 本発明の核酸抽出／増幅試薬に用いる添加剤の上限濃度２
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添加剤の濃度を検討した。
（１）参考例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に相当するもの）を、注射用水を用いて１０^４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。
反応液の組成：濃度は抽出液添加後（３０μＬ中）の最終濃度
６０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ８．６５）
各０．２５ｍＭ ｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰ、ｄＴＴＰ
各２．７ｍＭ ＡＴＰ、ＣＴＰ、ＵＴＰ、ＧＴＰ
３．０６ｍＭ ＩＴＰ
６０ｍＭ トレハロース
９．１Ｕ ＡＭＶ逆転写酵素
１４２Ｕ Ｔ７ ＲＮＡポリメラーゼ
表３に記載の濃度のＩＮＡＦプローブ（参考例２で調製）（配列番号４および５）
６．８％ シクロデキストリン
表３に記載の濃度の第一のプライマー
表３に記載の濃度の第二のプライマー
表６に記載の各種濃度の添加剤。

Example 2 Upper limit concentration of additive used for nucleic acid extraction / amplification reagent of the present invention 2
The concentration of the additive added to the nucleic acid extraction / amplification reagent of the present invention was examined by the following method.
(1) A type A (H1N1 subtype) influenza virus standard RNA (corresponding to SEQ ID NO: 1) prepared in Reference Example 1 is diluted with water for injection to 10 ⁴ copies / 5 μL, and an RNA sample Used as.
(2) 15 μL of a reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).
Composition of reaction solution: Concentration is 60 mM Tris-HCl buffer (pH 8.65) at final concentration after addition of extract (in 30 μL)
0.25 mM dATP, dCTP, dGTP, dTTP each
2.7 mM each ATP, CTP, UTP, GTP
3.06 mM ITP
60 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase The concentration of the INAF probe described in Table 3 (prepared in Reference Example 2) (SEQ ID NOs: 4 and 5)
6.8% Cyclodextrin First primer at a concentration described in Table 3 Second primer at a concentration described in Table 3 Additives of various concentrations described in Table 6.

(3) After incubating the above reaction solution at 46 ° C. for 4 minutes, 15 μL of an extract solution having the following composition was added. To the extract, 1.5 μL of the RNA sample was added in advance.
Composition of extract: Concentration is 22.2 mM magnesium chloride 35 mM potassium chloride after addition of extract (in 30 μL)
1.2% Glycerol 10.3% DMSO
0.05% Tween20
1.5% sodium cholate (4) Detection was performed in the same manner as in Example 1. The results are shown in Table 6.

実施例３ 本発明の核酸抽出／増幅試薬に用いる添加剤の検討
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添加剤を検討した。表７に示す添加剤は（２）もしくは（３）に示す反応液や抽出液のいずれかに添加した。
（１）参考例３で入手した鼻腔擦過物が付着したスワブを、１３６μＬの注射用水に懸濁した。当該懸濁液を、臨床検体として用いた。臨床検体は２人分使用した。イムノクロマト法ではいずれもＡ型であった。

Example 3 Examination of Additives Used in Nucleic Acid Extraction / Amplification Reagent of the Present Invention Additives added to the nucleic acid extraction / amplification reagent of the present invention were examined by the following method. The additives shown in Table 7 were added to either the reaction solution or the extraction solution shown in (2) or (3).
(1) The swab to which the nasal scrape obtained in Reference Example 3 was adhered was suspended in 136 μL of water for injection. The suspension was used as a clinical specimen. Two clinical samples were used. In the immunochromatography method, all were A type.

(2) 15 μL of a reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).
Composition of reaction solution: Concentration is 60 mM Tris-HCl buffer (pH 8.65) at final concentration after addition of extract (in 30 μL)
0.25 mM dATP, dCTP, dGTP, dTTP each
2.7 mM each ATP, CTP, UTP, GTP
3.06 mM ITP
60 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase The concentration of the INAF probe described in Table 3 (prepared in Reference Example 2) (SEQ ID NOs: 4 and 5)
6.8% cyclodextrin First primer at the concentration described in Table 3 Second primer at the concentration described in Table 3.

(3) After incubating the above reaction solution at 46 ° C. for 4 minutes, 15 μL of an extract solution having the following composition was added. 2 μL of the clinical specimen was added to the extract in advance.
Composition of extract: Concentration is 22.2 mM magnesium chloride 35 mM potassium chloride after addition of extract (in 30 μL)
1.2% Glycerol 10.3% DMSO
0.05% Tween20
1.5% sodium cholate.

  (4) Detection was performed in the same manner as in Example 1. The results are shown in Table 7. Both additives had a faster detection time than when water for injection was added. It was also found that the addition of a reducing agent has an effect of avoiding TRC reaction inhibition by the specimen.

実施例４ 還元剤の効果１
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還元剤の臨床検体によるＴＲＣ反応阻害回避効果を調べた。
（１）参考例３で入手した鼻腔擦過物が付着したスワブを、１３６μＬの注射用水に懸濁した。当該懸濁液を、臨床検体として用いた。イムノクロマト法ではＡ型であった。

Example 4 Effect 1 of reducing agent
By the following method, the TRC reaction inhibition avoidance effect by the clinical sample of the reducing agent added to the nucleic acid extraction / amplification reagent of the present invention was examined.
(1) The swab to which the nasal scrape obtained in Reference Example 3 was adhered was suspended in 136 μL of water for injection. The suspension was used as a clinical specimen. The immunochromatography method was type A.

(2) 15 μL of a reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).
Composition of reaction solution: Concentration is 60 mM Tris-HCl buffer (pH 8.65) at final concentration after addition of extract (in 30 μL)
0.25 mM dATP, dCTP, dGTP, dTTP each
2.7 mM each ATP, CTP, UTP, GTP
3.06 mM ITP
60 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase The concentration of the INAF probe described in Table 3 (prepared in Reference Example 2) (SEQ ID NOs: 4 and 5)
6.8% cyclodextrin First primer at the concentration described in Table 3 Second primer at the concentration described in Table 3.

(3) After incubating the above reaction solution at 46 ° C. for 4 minutes, 15 μL of an extract solution having the following composition was added. To the extract, 1 μL, 2 μL, 3 μL, or 4 μL of the clinical specimen was added in advance.
Composition of extract: Concentration is 22.2 mM magnesium chloride 35 mM potassium chloride after addition of extract (in 30 μL)
1.2% Glycerol 10.3% DMSO
0.05% Tween20
1.5% aqueous sodium cholate 5 mM DTT.

  (4) Detection was performed in the same manner as in Comparative Example 1. The results are shown in Table 8. In any case, it was found that the detection time was greatly improved when DTT was added.

実施例５ 還元剤の効果２
より安定性の高い還元剤であるＴＣＥＰでの検出を試みた。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還元剤の臨床検体によるＴＲＣ反応阻害回避効果を調べた。
（１）参考例３で入手した鼻腔擦過物が付着したスワブを、１３６μＬの注射用水に懸濁した。当該懸濁液を、臨床検体として用いた。イムノクロマト法ではＡ型であった。

Example 5 Effect 2 of reducing agent
Detection was attempted with TCEP, which is a more stable reducing agent. By the following method, the TRC reaction inhibition avoidance effect by the clinical sample of the reducing agent added to the nucleic acid extraction / amplification reagent of the present invention was examined.
(1) The swab to which the nasal scrape obtained in Reference Example 3 was adhered was suspended in 136 μL of water for injection. The suspension was used as a clinical specimen. The immunochromatography method was type A.

(2) 15 μL of a reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).
Composition of reaction solution: Concentration is 60 mM Tris-HCl buffer (pH 8.65) at final concentration after addition of extract (in 30 μL)
0.25 mM dATP, dCTP, dGTP, dTTP each
2.7 mM each ATP, CTP, UTP, GTP
3.06 mM ITP
60 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase The concentration of the INAF probe described in Table 3 (prepared in Reference Example 2) (SEQ ID NOs: 4 and 5)
6.8% cyclodextrin First primer at the concentration described in Table 3 Second primer at the concentration described in Table 3.

(3) After incubating the above reaction solution at 46 ° C. for 4 minutes, 15 μL of an extract solution having the following composition was added. The extract was preliminarily added with 4 μL of the clinical specimen.
Composition of extract: Concentration is 22.2 mM magnesium chloride 35 mM potassium chloride after addition of extract (in 30 μL)
1.2% Glycerol 10.3% DMSO
0.05% Tween20
1.5% sodium cholate aqueous solution 0.1mM TCEP
0.2 mM KOH.

  (4) Detection was performed in the same manner as in Comparative Example 1. The results are shown in Table 9. It was found that the detection time was improved when TCEP was added. Sensitivity was worse than DTT.

実施例６ ＴＣＥＰによる検出の高感度化１
ＴＣＥＰを添加することで、抽出液のｐＨが変化し、ＴＲＣ反応に影響を及ぼしている可能性が考えられた。そこで、ｐＨ調整剤を抽出液に加え、あらかじめｐＨを調製する以下の方法により、本発明の核酸抽出／増幅試薬に添加するＴＣＥＰの臨床検体によるＴＲＣ反応阻害回避効果を調べた。
（１）参考例３で入手した鼻腔擦過物が付着したスワブを、１３６μＬの注射用水に懸濁した。当該懸濁液を、臨床検体として用いた。イムノクロマト法ではＡ型であった。

Example 6 Sensitivity enhancement by TCEP 1
By adding TCEP, it was considered that the pH of the extract was changed and had an influence on the TRC reaction. Therefore, the effect of avoiding the inhibition of TRC reaction by the clinical specimen of TCEP added to the nucleic acid extraction / amplification reagent of the present invention was examined by the following method of adding a pH adjuster to the extract and adjusting the pH in advance.
(1) The swab to which the nasal scrape obtained in Reference Example 3 was adhered was suspended in 136 μL of water for injection. The suspension was used as a clinical specimen. The immunochromatography method was type A.

(2) 15 μL of a reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).
Composition of reaction solution: Concentration is final concentration after addition of extract (in 30 μL) Tris-HCl buffer (pH 8.65)
0.25 mM dATP, dCTP, dGTP, dTTP each
2.7 mM each ATP, CTP, UTP, GTP
3.06 mM ITP
60 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase The concentration of the INAF probe described in Table 3 (prepared in Reference Example 2) (SEQ ID NOs: 4 and 5)
6.8% cyclodextrin First primer at the concentration described in Table 3 Second primer at the concentration described in Table 3.

(3) After incubating the above reaction solution at 46 ° C. for 4 minutes, 15 μL of an extract solution having the following composition was added. 2 μL of each of the clinical specimens was previously added to the extract.
Composition of extract: Concentration is final concentration after addition of extract (in 30 μL) Tris-HCl buffer (pH 8.65)
22.2 mM magnesium chloride 35 mM potassium chloride
1.2% Glycerol 10.3% DMSO
0.05% Tween20
1.5% aqueous sodium cholate 0.1 mM TCEP.

  (4) Detection was performed in the same manner as in Comparative Example 1. The results are shown in Table 10. In this clinical specimen, because the inhibition was very strong, the Tris-HCl buffer solution (pH 8.65) was added to the extract, although the Tris-HCl buffer solution was not added to the extract and could not be detected by TCEP alone. It was found that the detection time was greatly improved.

実施例７ ＴＣＥＰによる検出の高感度化２
ｐＨ調整剤を抽出液に加え、ＫＯＨによりあらかじめｐＨを調製する以下の方法により、本発明の核酸抽出／増幅試薬に添加するＴＣＥＰの臨床検体によるＴＲＣ反応阻害回避効果を調べた。
（１）参考例３で入手した鼻腔擦過物が付着したスワブを、１３６μＬの注射用水に懸濁した。当該懸濁液を、臨床検体として用いた。イムノクロマト法ではＡ型であった。

Example 7 Higher detection sensitivity by TCEP 2
The effect of avoiding the inhibition of TRC reaction by a clinical sample of TCEP added to the nucleic acid extraction / amplification reagent of the present invention was examined by the following method in which a pH adjusting agent was added to the extract and the pH was previously adjusted with KOH.
(1) The swab to which the nasal scrape obtained in Reference Example 3 was adhered was suspended in 136 μL of water for injection. The suspension was used as a clinical specimen. The immunochromatography method was type A.

(2) A reagent prepared by freeze-drying a reaction solution having the following composition in a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI) was prepared.
Composition of reaction solution: Concentration is 6 mM Tris-HCl buffer (pH 8.65) at final concentration after addition of extract (in 30 μL)
0.25 mM dATP, dCTP, dGTP, dTTP each
2.7 mM each ATP, CTP, UTP, GTP
3.06 mM ITP
198 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase INAF probe at concentration shown in Table 3 (prepared in Reference Example 2) (SEQ ID NOs: 4 and 5)
6.8% cyclodextrin First primer at the concentration described in Table 3 Second primer at the concentration described in Table 3.

(3) After incubating the above reaction solution at 46 ° C. for 4 minutes, 30 μL of an extract solution having the following composition was added. The extract was preliminarily added with 4 μL of the clinical specimen. As the KOH and DMSO concentrations, concentrations optimized for lyophilized products were used.
Composition of extract: Concentration is 54 mM Tris-HCl buffer (pH 8.65) after addition of extract (in 30 μL)
22.2 mM magnesium chloride 57.5 mM potassium chloride
1.2% Glycerol 11% DMSO
0.05% Tween20
1.5% sodium cholate aqueous solution 4mM TCEP
Each concentration KOH.

  (4) Detection was performed in the same manner as in Comparative Example 1. The results are shown in Table 11. The detection time was significantly improved when KOH was added as well as the Tris-HCl buffer (pH 8.65).

実施例８ 複数の添加剤を添加した場合の効果
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添加剤を複数添加した場合の臨床検体によるＴＲＣ反応阻害回避効果を調べた。
（１）参考例３で入手した鼻腔擦過物が付着したスワブを、１３６μＬの注射用水に懸濁した。当該懸濁液を、臨床検体として用いた。イムノクロマト法ではＡ型であった。

Example 8 Effect of Adding Multiple Additives The TRC reaction inhibition avoidance effect by clinical specimens when multiple additives to be added to the nucleic acid extraction / amplification reagent of the present invention were added was examined by the following method.
(1) The swab to which the nasal scrape obtained in Reference Example 3 was adhered was suspended in 136 μL of water for injection. The suspension was used as a clinical specimen. The immunochromatography method was type A.

(2) 15 μL of a reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).
Composition of reaction solution: Concentration is 6 mM Tris-HCl buffer (pH 8.65) at final concentration after addition of extract (in 30 μL)
0.25 mM dATP, dCTP, dGTP, dTTP each
2.7 mM each ATP, CTP, UTP, GTP
3.06 mM ITP
60 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase The concentration of the INAF probe described in Table 3 (prepared in Reference Example 2) (SEQ ID NOs: 4 and 5)
6.8% cyclodextrin First primer at the concentration described in Table 3 Second primer at the concentration described in Table 3.

(3) After incubating the above reaction solution at 46 ° C. for 4 minutes, 15 μL of an extract solution having the following composition was added. 2 μL of each of the clinical specimens was previously added to the extract.
Composition of extract: Concentration is final concentration after addition of extract (in 30 μL) Tris-HCl buffer (pH 8.65)
22.2 mM magnesium chloride 35 mM potassium chloride
1.2% Glycerol 10.3% DMSO
0.05% Tween20
1.5% sodium cholate aqueous solution 2mM TCEP
Various concentrations of additives listed in Table 12.

  (4) Detection was performed in the same manner as in Comparative Example 1. The results are shown in Table 12. Even if a polysaccharide-degrading enzyme or an antioxidant was contained in the solution containing TCEP, the sensitivity was not affected. Considering that only the polysaccharide degrading enzyme had the effect of avoiding the inhibition of the TRC reaction by the specimen, it was found that there is no problem even if a plurality of additives that do not have the stronger avoidance effect than TCEP are included.

Claims (5)

In a viral nucleic acid extraction and amplification kit having i) a viral nucleic acid extraction reagent, and ii) a viral nucleic acid amplification reagent,
containing a reducing agent in i) viral nucleic acid extraction reagent or ii) viral nucleic acid amplification reagent,
And the kit for amplifying a viral nucleic acid without purification after extraction. The kit according to claim 1, wherein i) viral nucleic acid extraction reagent or ii) viral nucleic acid amplification reagent contains a polysaccharide degrading enzyme and / or a dummy nucleic acid. The kit according to claim 1 or 2, wherein the virus is an influenza virus. The kit according to any one of claims 1 to 3, wherein the reducing agent is dithiothreitol or tris (2carboxyethyl) phosphine. A sample containing a virus is brought into contact with i) a virus nucleic acid extraction reagent and ii) a virus nucleic acid amplification reagent of the kit according to any one of claims 1 to 4, and the virus nucleic acid is amplified without being purified after extraction. A method for extracting and amplifying viral nucleic acid.