JP2018166410A - Method for long-term storage of nucleic acids in biological samples - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for stably storing nucleic acid contained in cells, bacteria, viruses and the like in a biological sample for a long period of time. The sample using the above method can be used in a step of concentrating cells, bacteria, viruses, etc., and a culturing step. A method for storing nucleic acid contained in cells, bacteria, viruses, etc. in a biological sample, which comprises a step of mixing the biological sample with a reducing agent, wherein the reducing agent is dithiothreitol. One or more selected from the group consisting of dithiothreitol, mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl), N-acetyl-L-cysteine (NALC), and L-cysteine hydrochloride. The above-mentioned problem is solved by the method of storing nucleic acid including. [Selection diagram] None

Description

  The present invention relates to a method for stably storing and detecting a nucleic acid contained in cells, bacteria or viruses in a biological sample for a long period of time, and a reagent for storage.

  In the fields of clinical tests, public health, and food tests, tests for measuring DNA or RNA (target nucleic acid) such as cells, bacteria, fungi, and viruses contained in a sample are performed. Currently, many nucleic acid amplification reagents for measuring one or more target nucleic acids are available. In general, since a target nucleic acid in a sample is very small, a nucleic acid amplification reaction of the target nucleic acid is usually performed for the test. Various methods are used for these nucleic acid amplification reactions. For example, the PCR method (see Patent Documents 1, 2, and 3), the LAMP method (see Non-Patent Document 1), the RT-PCR method, the NASBA method ( Patent Documents 4 and 5), TMA method (see Patent Document 6), TRC method (see Patent Document 7 and Non-Patent Document 2), and the like. In the nucleic acid amplification test, biological samples such as body fluid, digestive fluid, urine, feces, saliva, semen, vaginal fluid, scraped material, and culture fluid are used. Usually, these samples contain a nucleolytic enzyme and a proteolytic enzyme, and it is difficult to detect the nucleic acid from a sample in which a bacterial or viral nucleic acid is stored in a biological sample for a long period of time. In particular, urine samples contain an enzyme that degrades nucleic acids and substances that inhibit urine-specific nucleic acid amplification reactions, and it is usually difficult to use urine itself for amplification reactions. However, it is difficult to immediately perform the above-described inspection after obtaining a biological sample because of the need for equipment investment, an increase in inspection costs, and a shortage of inspectors.

  Therefore, a method for stably storing a nucleic acid in a sample for nucleic acid amplification test is required when performing the above test.

  Commonly used as a preservation solution for a sample for nucleic acid amplification test is a guanidine solution or a solution containing a surfactant. However, there is a problem that these chemical substances become reaction inhibitory substances in the nucleic acid amplification step or the nucleic acid purification step often performed before the nucleic acid amplification test. In addition, these chemical substances have protein-denaturing action, and also have the action of altering the membrane structure of bacteria and viruses and releasing nucleic acids, so the process of concentrating cells, bacteria and viruses from biological samples, and the process for culturing There is a problem that it cannot be used.

  Until now, the use of reducing agents includes stabilization of enzyme reactions and protein stabilization, but nucleic acids can be stably detected from biological samples used in the present invention, especially cells, bacteria, viruses, etc. contained in urine. There are no reports used to do this.

US Pat. No. 4,683,195 U.S. Pat.No. 4,683,202 US Pat. No. 4,965,188 Japanese Patent No. 2650159 Japanese Patent No. 3152927 Japanese Patent No. 3241717 JP 2000-14400 A

Thai H.H. T.A. C. et al. J. et al. Clin. Microbiol. 42, 1956-61 (2004) Ishiguro T. et al. Anal. Biochem. 314, 77-86 (2003)

  An object of the present invention is to provide a method for stably storing nucleic acids contained in cells, bacteria, viruses or the like in a biological sample, a method for detecting nucleic acids stored by the method, and a method for stabilizing nucleic acids. It is to provide a reagent.

As a result of intensive studies in view of the above-mentioned object, the present inventors have found that a nucleic acid in a biological sample can be stably stored by mixing a specific reducing agent with the biological sample, and the present invention has been completed. .

That is, this invention can be illustrated as follows.
[1] A method for preserving nucleic acid contained in cells, bacteria, viruses or the like in a biological sample, comprising the step of mixing the biological sample and a reducing agent, wherein the reducing agent is dithiothreitol, dithiol Contains one or more selected from the group consisting of erythritol, mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl), N-acetyl-L-cysteine (NALC), and L-cysteine hydrochloride A method for storing nucleic acids.
[2] The nucleic acid according to [1], wherein the cells, bacteria, viruses or the like in the biological sample are bacteria selected from the group consisting of bacteria belonging to the family Chlamydiaceae, bacteria belonging to the genus Neisseria, and mixtures thereof. How to save.
[3] A method for detecting nucleic acids contained in cells, bacteria, viruses, etc. in a biological sample stored by the storage method according to [1] or [2],
(1) A method for detecting a nucleic acid, comprising a step of extracting nucleic acid from the stored biological sample, and (2) a step of amplifying and detecting the nucleic acid extracted in step (1).
[4] Before the step (1), the method further includes a step of concentrating cells, bacteria or viruses in the biological sample, and the step (1) extracts a nucleic acid from the concentrated biological sample. The method according to [3], wherein
[5] The method for detecting a nucleic acid according to [3] or [4], wherein the step of amplifying and detecting the nucleic acid is performed by real-time monitoring of an indicator of the amount of the nucleic acid amplification product simultaneously with the nucleic acid amplification.
[6] In the step of amplifying and detecting the nucleic acid, the nucleic acid amplification is performed by a method selected from a PCR method, a NASBA method, a TMA method, an SDA method, a LAMP method, and a TRC method. The method for detecting a nucleic acid according to any one of [3] to [5].
[7] A reagent for preserving nucleic acid contained in cells, bacteria or viruses in a biological sample, which is dithiothreitol, dithioerythritol, mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride A reagent comprising one or more selected from the group consisting of (TCEP-HCl), N-acetyl-L-cysteine (NALC), and L-cysteine hydrochloride.

  The present invention provides methods and reagents for stably storing nucleic acids in biological samples. The method and reagent can be used in a method for detecting a nucleic acid in a biological sample. According to the present invention, nucleic acids in a biological sample can be easily detected in a stable state even at a refrigeration temperature or higher. Further, by using the present invention, it is possible to preserve without destroying the membrane structure of cells, bacteria or viruses contained in the biological sample, so that the cells, bacteria or viruses contained in the biological sample are concentrated. It can use for the process to perform and the process to culture | cultivate.

  Hereinafter, the present invention will be described in detail.

  One embodiment of the present invention is a method for stably preserving nucleic acids contained in cells, bacteria, viruses, or the like in a biological sample. Examples of the biological sample include body fluids such as urine, blood, and gargle, but are not particularly limited.

  Examples of the reducing agent include dithiothreitol, dithioerythritol, mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl), N-acetyl-L-cysteine (NALC), and L-cysteine hydrochloride mixture. Is exemplified, but is not particularly limited. The listed reducing agents may be used alone or in appropriate combination. Among the reducing agents, dithiothreitol or mercaptoethanol is preferable, but is not particularly limited. The concentration of the reducing agent is not particularly limited, but preferably 1 to 91 mM for dithiothreitol, 142 to 1284 mM for mercaptoethanol, more preferably 20 to 91 mM for dithiothreitol, and 142 to 286 mM for mercaptoethanol. .

Another aspect of the present invention is a nucleic acid contained in a cell, bacterium, virus, or the like in a biological sample stored by the method for stably preserving the nucleic acid contained in the cell, bacterium, virus, or the like in the biological sample. A method of detecting
There is provided a method for detecting a nucleic acid, comprising: (1) extracting a nucleic acid from the stored biological sample; and (2) amplifying and detecting the nucleic acid extracted in step (1).

  In the method for preserving cells, bacteria, viruses or the like in a biological sample according to an embodiment of the present invention, since a specific reducing agent is used, the membrane structure of the cells, bacteria, viruses or the like is not easily destroyed. . If the nucleic acid is released outside the membrane when the membrane structure is destroyed, it cannot be used for concentration or culture, but by using the preservation method of the present invention, cells, bacteria or viruses contained in the biological sample after storage. Etc. can be concentrated or cultured. The amount of nucleic acid used for detection can be increased by concentration or culture, and can be suitable for detection of nucleic acid from a small amount of sample. The means for increasing the amount of nucleic acid such as cells, bacteria or viruses before the nucleic acid detection step is not particularly limited, but concentration, particularly centrifugal concentration is preferred from the viewpoint of ease of operation.

  The method that can be employed in the nucleic acid amplification reaction is not particularly limited as long as it is a method for amplifying a nucleic acid, and examples thereof include a PCR method, a LAMP method, a NASBA method, a TMA method, an SDA method, and a TRC method.

  Examples of the bacteria contained in the biological sample used in the present invention include bacteria selected from the group consisting of bacteria belonging to the family Chlamydiaceae, bacteria belonging to the genus Neisseria, or mixtures thereof. Examples of the virus contained in the biological sample used in the present invention include a virus selected from the group consisting of adenovirus, cytomegalovirus, hepatitis C virus, hepatitis B virus or a mixture thereof. Examples of cells contained in the biological sample used in the present invention include, but are not limited to, epithelial cells infected with parasitic bacteria and viruses.

  Hereinafter, embodiments of the present invention will be described by way of examples. However, the present invention is not limited to these examples.

Example 1 Preparation of Oligonucleotide Labeled with Intercalating Fluorescent Dye Oligonucleotide probe labeled with an intercalating fluorescent dye (hereinafter referred to as INAF probe) shown in (A) below is disclosed in Japanese Patent Application Laid-Open No. 2000-316587. It was produced based on the method disclosed in No.
(A) Among oligonucleotides consisting of the nucleotide sequence described in SEQ ID NO: 1 (complementary sequence of nucleotide sequences from 856838 to 856856 of GenBank No. HE601794), the 10th cytosine from the 5 ′ end What labeled the pigment | dye as described in Formula (1) through the linker between 11th guanine.

Example 2 Detection test 1 of Chlamydia trachomatis nucleic acid contained in urine
A culture of 0.34 mL of Chlamydia trachomatis UW-3 / Cx strain (ATCC No. VR-885) was added to 100 mL of a pool of various urine specimens to prepare a sample.

  The prepared sample was dispensed by 10 mL, and 1 M dithiothreitol or 100% mercaptoethanol was added in the amount shown in Table 1, mixed well, and stored at 10 ° C.

  After elapse of the days shown in Table 3, the sample amount shown in Table 1 is added to a commercially available nucleic acid purification kit (TRCR nucleic acid purification kit, Tosoh Corp.), and the nucleic acid is added according to the instruction manual of the kit. Purified.

  The base sequences described in SEQ ID NOs: 1 to 4 are shown in Table 2.

The obtained purified product was recovered and subjected to a nucleic acid amplification reaction by the following method to verify whether nucleic acid was detected.
(1) A reaction solution having the following composition was dispensed into an evaporation drying tube and evaporated to dryness.
Composition of reaction solution: Concentration is as follows. Final concentration after addition of purified product and starting solution (in 30 μL) 60 mM Tris-HCl buffer (pH 8.6)
300 mM trehalose 0.25 mM each dATP, dCTP, dGTP, dTTP
2.6mM ATP, CTP, UTP each
1.9 mM GTP
3.1 mM ITP
0.16 μM Oligonucleotide for cleavage (modified hydroxyl group at 3 ′ end with amino group): SEQ ID NO: 2 (complementary sequence of nucleotide sequence from 856730 to 85649 of GenBank No. HE601794)
0.5 μM First primer: SEQ ID NO: 3 (T7 promoter added to 5 ′ end of nucleotide sequence from 856767 to 856791 of GenBank No. HE601794)
0.5 μM Second primer: SEQ ID NO: 4 (complementary sequence of nucleotide sequence from 856965th to 856986th of GenBank No. HE601794)
40 nM INAF probe (prepared in Example 1)
0.025mg / mL Bovine Serum Albumin 142U T7 RNA Polymerase 6.4U AMV Reverse Transcriptase (2) After the evaporative drying, 15 μL of the purified product is added, and then kept at 46 ° C. for 5 minutes. 15 μL of the solution was added and stirred.
Composition of the starting solution: final concentration at the time of reaction (in 30 μL) 11% dimethyl sulfoxide 20 mM magnesium chloride 85 mM potassium chloride 2% glycerol At the same time, the fluorescence intensity of the reaction solution was measured over time for 30 minutes. When the start liquid is added and stirring is completed, the reaction time is 0 minutes, and the reaction mixture's fluorescence intensity ratio (the fluorescence intensity value for a given time divided by the background fluorescence intensity ratio) exceeds 1.28. And the time at that time was defined as a positive time. The results are shown in Table 3. “ND” in Table 3 means that the fluorescence intensity ratio 30 minutes after the start of the reaction was 1.28 or less (negative determination).

  From the results in Table 3, nucleic acid was not detected after 20 days in the system in which the reducing agent was not mixed, but nucleic acid was detected in the system in which the reducing agent was mixed. From this, it is considered that a reducing agent is effective for stably detecting Chlamydia trachomatis nucleic acid in urine.

Example 3 Detection test 2 of Chlamydia trachomatis nucleic acid contained in urine
A 0.80 mL culture of Chlamydia trachomatis UW-3 / Cx strain (ATCC No. VR-885) was added to 80 mL of a pool of various urine specimens to prepare a sample.

  To 0.5 mL of the prepared sample, 1 M dithiothreitol or 5% sodium lauryl sulfate (Comparative Example) was added at the quantitative ratio shown in Table 4, and mixed well and stored at 10 ° C. .

  After the number of days shown in Table 5 has elapsed, the sample amount shown in Table 4 is collected and added to a commercially available nucleic acid purification kit (TRCR nucleic acid purification kit, Tosoh Corp.). Was purified.

  The obtained purified product was recovered and subjected to nucleic acid amplification reaction in the same manner as in Example 2 to verify whether nucleic acid was detected. The time when the start solution was added and the stirring was completed was set to 0 minute, and 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 1.28. A positive determination was made, and the time at that time was defined as a positive time. The results are shown in Table 5. “ND” in Table 5 means that the fluorescence intensity ratio 10 minutes after the start of the reaction was 1.28 or less (negative determination).

  From the results shown in Table 5, the nucleic acid was not detected after 1 day in the system mixed with sodium lauryl sulfate, and the nucleic acid was sometimes not detected after 7 days in the system without addition. In the system in which dithiothreitol was mixed, all nucleic acids were detected even after 11 days. From this, it is considered that a reducing agent is effective for stably detecting Chlamydia trachomatis nucleic acid in urine.

Example 4 Detection test 3 of Chlamydia trachomatis nucleic acid contained in urine
A 0.70 mL culture of Chlamydia trachomatis UW-3 / Cx strain (ATCC No. VR-885) was added to 210 mL of a pool of various urine specimens to prepare a sample.

  10 mL of the prepared sample as it is is added to a commercially available nucleic acid purification kit (TRCR nucleic acid purification kit, Tosoh Corp.), and 10 mL is a high-speed cooling centrifuge (model number MX-) at a strength of 1000 to 6000 × G (301, TOMY Seiko), the supernatant and the precipitate were separated, and the precipitate and the supernatant were added to the nucleic acid purification kit. In addition, 10 mL of the sample prepared in the same manner was prepared by adding 1.0 mL of 1 M dithiothreitol, and the same as above, added to a commercially available nucleic acid purification kit, 11 mL Centrifugation (including urine specimen, Chlamydia trachomatis culture solution and dithiothreitol) was prepared by subjecting the supernatant and precipitate separated to the nucleic acid purification kit.

  Each obtained sample was purified according to the instruction manual of the kit.

  The purified product was collected and subjected to nucleic acid amplification reaction in the same manner as in Example 2 to verify whether nucleic acid was detected. The time when the start solution was added and the stirring was completed was set to 0 minute, and 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 1.28. A positive determination was made, and the time at that time was defined as a positive time. The results are shown in Table 6. “ND” in Table 6 means that the fluorescence intensity ratio 10 minutes after the start of the reaction was 1.28 or less (negative determination).

  From the results shown in Table 6, no nucleic acid was detected after 4 days in the additive-free system, and no nucleic acid was detected after 28 days even after centrifugal concentration. On the other hand, in the system in which dithiothreitol was mixed, all nucleic acids were detected after 28 days in both the centrifugally concentrated system and the non-centrifugated system. Thus, it is possible to use centrifugal concentration by using the above method, and it is considered that a reducing agent is effective for stably detecting Chlamydia trachomatis nucleic acid in urine.

Claims (7)

A method for preserving nucleic acids contained in cells, bacteria or viruses in a biological sample,
Mixing the biological sample with a reducing agent,
The reducing agent is dithiothreitol, dithioerythritol, mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl), N-acetyl-L-cysteine (NALC), and L-cysteine hydrochloride. A method for storing a nucleic acid, comprising at least one selected from the group consisting of: The method for preserving a nucleic acid according to claim 1, wherein the cells, bacteria, or viruses in the biological sample are bacteria selected from the group consisting of bacteria belonging to the family Chlamydiaceae, bacteria belonging to the genus Neisseria, and mixtures thereof. . A method for detecting nucleic acids contained in cells, bacteria, viruses, or the like in a biological sample stored by the storage method according to claim 1 or 2,
(1) A method for detecting a nucleic acid, comprising a step of extracting nucleic acid from the stored biological sample, and (2) a step of amplifying and detecting the nucleic acid extracted in step (1). The step (1) further includes a step of concentrating cells, bacteria or viruses in the biological sample, and the step (1) is a step of extracting nucleic acid from the concentrated biological sample. The method according to claim 3. The method for detecting a nucleic acid according to claim 3 or 4, wherein the step of amplifying and detecting the nucleic acid is performed by real-time monitoring of an indicator of the amount of the nucleic acid amplification product simultaneously with the nucleic acid amplification. The nucleic acid amplification in the step of amplifying and detecting the nucleic acid is performed by a method selected from any of a PCR method, a NASBA method, a TMA method, an SDA method, a LAMP method, or a TRC method. The method for detecting a nucleic acid according to any one of 3 to 5. A reagent for preserving nucleic acid contained in cells, bacteria or viruses in a biological sample, which is dithiothreitol, dithioerythritol, mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride (TCEP- HCl), N-acetyl-L-cysteine (NALC), and a reagent comprising one or more selected from the group consisting of L-cysteine hydrochloride.