JP2019129798A - Quick and efficient isothermal amplification reaction using heating sample - Google Patents

Abstract

To provide a method for amplifying and detecting a target nucleic acid by a quick and efficient isothermal amplification reaction.SOLUTION: The present invention provides a method for amplifying and detecting a target nucleic acid, whereby, in an isothermal amplification reaction, a sample is added at a temperature higher than the temperature at which the isothermal amplification reaction is performed.SELECTED DRAWING: None

Description

  The present invention relates to a method for amplifying and detecting a target nucleic acid using an efficient isothermal amplification reaction using a heated sample.

  As a method for amplifying a nucleic acid, a polymerase chain reaction method (PCR method) is widely known. This method repeats a cycle consisting of heat denaturation-primer annealing-extension reaction in the presence of a pair of primers having a sequence complementary to or homologous to a part of a specific base sequence in the target DNA and a thermostable DNA polymerase. It is a method of amplifying DNA by performing. In addition, when the target nucleic acid is RNA, a so-called RT-PCR method is known in which a PCR method is performed after cDNA is once synthesized by reverse transcriptase. However, the PCR method and the RT-PCR method need to raise and lower the reaction temperature a number of times rapidly, which has been a barrier for labor saving and cost reduction of the reaction apparatus at the time of automation.

  On the other hand, methods such as NASBA method (Patent Documents 1 and 2), TMA method (Patent Document 3), and TRC method (Patent Document 4 and Non-Patent Document 1) are known as methods for amplifying nucleic acids at a constant temperature using RNA as a target. ing. Since these methods are isothermal amplification reactions in which nucleic acids are amplified at a constant temperature without raising or lowering the reaction temperature, nucleic acids can be easily amplified. Therefore, it can be said that it is a preferable method from the viewpoint of labor saving and cost reduction of the reaction apparatus at the time of automation.

  In the clinical laboratory field of infectious diseases, it is important to detect pathogens simply, quickly and with high sensitivity. As a method for detecting a pathogen with high sensitivity, a detection method using a nucleic acid amplification method is known. However, these nucleic acid amplification reactions are required to obtain results in a shorter time in the clinical laboratory field, which is often urgent.

Japanese Patent No. 2650159 Japanese Patent No. 3152927 Japanese Patent No. 3241717 JP 2000-014400 A

Ishiguro, T .; et al, Analytical Biochemistry, 314, 77-86 (2003).

  An object of the present invention is to provide a method for amplifying and detecting a target nucleic acid by a more rapid and efficient isothermal amplification reaction.

As a result of intensive studies to solve the above problems, the present inventors have completed the present invention. That is, the present invention includes the following aspects (1) to (5).
(1) In the isothermal amplification reaction, the temperature higher than that of the method (2) for amplifying and detecting the target nucleic acid, including the step of adding to the reaction reagent a sample having a temperature higher than the temperature at which the isothermal amplification reaction is performed is 0.8 ° C The method described in (1) above.
(3) The method according to (1), wherein the higher temperature is 3.4 ° C. or higher.
(4) The method according to (1), wherein the higher temperature is 9.9 ° C. or higher.
(5) The method according to any one of (1) to (4), wherein the reaction reagent is in a dry state.

  Hereinafter, the present invention will be described in detail.

  In one embodiment of the present invention, the isothermal amplification reaction may be a nucleic acid amplification reaction that is performed isothermally. For example, a first primer having a sequence homologous to part of a viral nucleic acid and part of the viral nucleic acid. A second primer having a complementary sequence, an enzyme having RNA-dependent DNA polymerase activity, an enzyme having DNA-dependent DNA polymerase activity, an enzyme having ribonuclease H (RNase H) activity, and RNA polymerase activity Wherein either of the first primer and the second primer corresponds to the enzyme having the RNA polymerase activity on the 5 ′ end side thereof. Examples of the method for adding the promoter sequence (for example, TMA method, TRC method, NASBA method).

  In one embodiment of the present invention, a temperature suitable for each isothermal amplification reaction can be appropriately selected as the temperature at which the isothermal amplification reaction is performed. For example, in the TRC method, it is selected from 43 ° C. or 46 ° C. depending on the heat resistance of the enzyme used.

  The higher temperature sample in one embodiment of the present invention may be a temperature higher than the temperature at which the isothermal amplification reaction is performed, for example, 0.8 ° C. or higher, preferably 3.4 ° C. or higher, more preferably 9 A sample heated to a liquid temperature higher than 9 ° C. Any heating method may be used, and heating by a general heat block can be exemplified.

  In one embodiment of the present invention, the nucleic acid to be detected may be DNA or RNA. For example, RNA may be selected in the TRC method. The nucleic acid may be DNA or RNA itself, and may be a microorganism, cell, virus, or the like containing DNA or RNA. As an example of a sample containing nucleic acid in one embodiment of the present invention, preferably 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 sample containing nucleic acid in the extraction reagent of the present invention.

  In one embodiment of the present invention, the sample only needs to include a sample containing the nucleic acid to be detected. For example, RNA, DNA, microorganisms, cells, viruses, etc. are simply dissolved in water, physiological saline, or a buffer solution. And biological samples containing microorganisms, cells, viruses, and the like, filter papers containing the biological samples, swabs, 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, tears, It is not limited to these. The sample may further contain a surfactant, a denaturant, an organic solvent, etc. as components necessary for nucleic acid extraction. Components necessary for isothermal amplification reaction, for example, salts such as magnesium salt and potassium salt, trehalose, etc. Sugars, sugar alcohols such as glycerol, nucleic acid components, organic solvents, nucleic acids such as primers and probes, buffer solution components, and the like.

  The reaction reagent in one embodiment of the present invention includes components necessary for isothermal amplification reaction, 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. In addition, nucleic acids such as primers and probes, buffer components, and the like can be contained, and together with the sample containing the nucleic acids, the final concentration is adjusted to a necessary level.

  The reaction reagent in one embodiment of the present invention may be in a dry form. Since the reaction reagent contains an enzyme, when it is in a dry state, the storage period can be extended as compared with the liquid state, and the temperature suitable for storage can be made closer to room temperature. Furthermore, there exists an advantage that it is easy to operate when transporting and using it by making it dry.

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

  According to the present invention, by using a sample having a temperature higher than the amplification temperature, the isothermal amplification reaction can be carried out more rapidly and efficiently than when the sample is not heated to a high temperature.

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

Example 1 Preparation of Standard RNA Primers and intercalator fluorescent dye standard nucleic acid probes used in Examples described later were prepared by the method described in JP-A-2006-131498.

Example 2 Preparation Method of Evaporative Drying Amplification Reagent A 18.5 μL reagent solution having the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and a vacuum freeze dryer (Virtis Advantage Plus). (Manufactured by Central Scientific Trading Co., Ltd.) at 25 ° C., 100 torr for 16 hours, 50 ° C., 1.5 hours, and 25 ° C. until the inside temperature settled down. The amplification reagent after drying was sealed and stored at 4 ° C. with a desiccant.

Composition of reagent solution: concentration is final concentration of TRC reaction (in 30 μL) 6 mM Tris-HCl (pH 8.65)
0.25 mM dATP, dCTP, dGTP, dTTP each
2.7 mM each ATP, CTP, UTP, GTP
3.06 mM ITP
198.9 mM trehalose 9.1 U AMV reverse transcriptase 142 U T7 RNA polymerase each 20 nM INAF probe (SEQ ID NO: 1; prepared in Example 1)
First primer (SEQ ID NO: 2; prepared in Example 1)
Second primer (SEQ ID NO: 3; prepared in Example 1)
8.2% (w / w) 2-hydroxypropyl-γ-cyclodextrin.

Example 3 Effect of Heated Sample The effect of the heated sample was examined by the following method.
(1) A virus sample was prepared by diluting influenza B virus (B / Massachettes / 2/2012 strain) to a concentration of 1.0 × 10 ⁻¹ TCID ₅₀ / ml with water for injection.
(2) 50 μL of the virus sample prepared in (1) was added to 1000 μL of a virus extract solution having the following composition and stirred to obtain a sample solution.

Composition of virus extract:
22.2 mM Magnesium chloride 75.0 mM Potassium chloride 1.2% Glycerol 11.0% DMSO
0.05% (v / v) Tween 20
1.5% (w / w) sodium cholate 54 mM Tris-HCl (pH 8.65)
4 mM Tris (2-carbethyl) phosphine Hydrochloride
3 mM KOH
(3) After heating the sample solution of (2) at the set temperature shown in Table 1 for 1 minute, 30 μL of the evaporated dry reagent prepared in Example 2 was added and mixed with a micropipette.
(4) Using a fluorescence spectrophotometer with 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 case where the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value for a predetermined time by the background fluorescence intensity ratio) exceeded 1.2 was determined as positive. Table 1 shows the detection time as the time required until a positive determination was obtained with the reagent mixing time of 0 minutes.
(5) Moreover, 1000 microliters of virus extracts prepared separately were heated similarly, and the liquid temperature of the sample was measured by measuring the liquid temperature.

  As a result, the detection time was 6.39 minutes when the sample temperature was about 46 ° C., the same as the reaction temperature, whereas it was 4.83 minutes when the sample temperature was higher, 46.8 ° C. The amplification reaction could be performed more quickly. Furthermore, this effect was improved as the liquid temperature was raised, and it reached 2.92 minutes at a liquid temperature of 55.9 ° C., which was about 10 ° C. higher than the reaction temperature, and then almost the same rapidity was exhibited up to 74.4 ° C. .

  That is, it was shown that by using a sample having a temperature higher than the amplification temperature, the isothermal amplification reaction can be carried out more quickly and efficiently than in the case where the temperature is not increased.

Claims (5)

In the isothermal amplification reaction, a method for amplifying and detecting a target nucleic acid, comprising adding a sample having a temperature higher than the temperature at which the isothermal amplification reaction is performed to a reaction reagent. The method of claim 1, wherein the higher temperature is 0.8 ° C or higher. The method of claim 1, wherein the higher temperature is 3.4 ° C or higher. The method of claim 1, wherein the higher temperature is 9.9 ° C or higher. The method according to claim 1, wherein the reaction reagent is in a dry state.