JP2017163904A - Method of stabilizing probe in nucleic acid detection reaction liquid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of inhibiting the degradation of a probe in a nucleic acid detection reaction liquid, in a nucleic acid detection method using the probe, and enables stable gene analysis.SOLUTION: The present invention provides a nucleic acid detection method that uses a probe and allows ammonium sulfate and/or tetramethylammonium acetate to coexist.SELECTED DRAWING: None

Description

The present invention relates to a method for stabilizing a nucleic acid detection probe (hereinafter abbreviated as “probe”) in a nucleic acid detection reaction solution in a molecular biological detection method such as detection of gene amplification rate or gene mutation.

Detection of genes and nucleic acids is an important operation method in molecular biology. Various methods for detecting nucleic acids have been developed so far, and probe hybridization methods and polymerase chain reaction (PCR) methods are relatively popular.

In PCR, in recent years, real-time PCR (hereinafter abbreviated as “qPCR”) capable of monitoring the production process of an amplification product over time due to high measurement sensitivity and rapidity has been widely implemented. There is a technique in which various fluorescent probes are used for main qPCR and PCR is combined with hybridization probing. As a qPCR using a probe, there are methods such as TaqMan (registered trademark) (see, for example, Non-Patent Document 1), Molecular Beacon, Hybridization Probe, Cycling Probe, etc., and amplification using a technique such as fluorescence energy transfer It is devised so that the fluorescence increases or decreases in correlation with the amount of nucleic acid.

For example, the TaqMan (registered trademark) probe method which is generally widely used is a method using a probe in which a fluorescent substance is bound to the 5 'end and a quencher is attached to the 3' end. This probe anneals to the template, but fluorescence is suppressed by the quencher even when it is irradiated with excitation light. When the complementary strand is extended, the probe is hydrolyzed by the 5 'to 3' exonuclease activity of the DNA polymerase, the fluorescent substance is released from the probe, and emits fluorescence by leaving the quencher. By detecting this fluorescence, an increase in fluorescence intensity due to amplification can be monitored.

In the nucleic acid detection method using a probe, when performing gene analysis of a large amount of sample such as HTS (High Throughput Screening), it is assumed that the reaction solution is left for a long time (several hours to several days) after preparation. However, according to the study by the inventors, it has become clear that the probe may be decomposed by leaving the reaction solution at room temperature. For example, in the TaqMan (registered trademark) probe method, a plurality of cases in which the Ct value is delayed or the detection itself becomes impossible by leaving the prepared reaction solution at room temperature were confirmed. For the reasons described above, it has been found as a new problem to stabilize the probe more effectively in the nucleic acid detection reaction solution.

The present inventors further considered the undetectable phenomenon in the TaqMan probe method. As a result, it was observed that the background fluorescence was abnormally increased in the above-mentioned non-detectable example, and this was caused by the hot start method (DNA polymerase at room temperature using an antibody against DNA polymerase in a nucleic acid detection reaction solution). DNA polymerase having 5 ′ → 3 ′ exonuclease activity is not completely suppressed by a technique for blocking the activity of DNA polymerase (see, for example, Non-Patent Document 2). We thought it was to disassemble the probe.
Based on the above considerations, the present inventors have conducted extensive research to solve the above problems, and as a result, a substance having a chelating action (hereinafter referred to as “chelating agent”) is allowed to coexist in the nucleic acid detection reaction solution. And found that the degradation of the probe can be suppressed. However, the above-described method alone may not sufficiently suppress the decomposition of the probe, and a new method for suppressing the decomposition of the probe is necessary.

Holland et al., Proc. Natl. Acad. Sci. 88, 1991, 7276-7280 Kellogg et al., Biotechniques, 16, 1994, 1134-1137.

An object of the present invention is to provide a novel method for suppressing the degradation of a probe in a nucleic acid detection reaction solution (hereinafter sometimes referred to as “reaction solution”) in a nucleic acid detection method using a probe. It is in.

As a result of intensive studies to solve the above problems, the present inventors have found that ammonium sulfate or tetramethylammonium acetate can coexist in a nucleic acid detection reaction solution to suppress probe degradation and complete the present invention. I came to let you.

That is, the present invention has the following configuration.
Item 1. A method for stabilizing a probe, characterized in that ammonium sulfate and / or tetramethylammonium acetate coexist in a nucleic acid detection reaction solution used in a nucleic acid detection method using a probe.
Item 2. Item 2. The method for stabilizing a probe according to Item 1, wherein ammonium sulfate and / or tetramethylammonium acetate coexist at a concentration of 1 mM to 40 mM.
Item 3. Item 3. The method according to Item 1 or 2, wherein the nucleic acid detection method is PCR using a probe.
Item 1 to 3, wherein the nucleic acid detection solution is stored for 24 hours under the temperature condition of Item 4.5 ° C to 35 ° C, and then satisfies the requirement of either (a) or (b) A method for stabilizing the probe according to 1.
(A) Ct value before storage / Ct value after storage> 0.8
(B) Fluorescence intensity at the beginning of the cycle before storage / Fluorescence intensity at the beginning of the cycle after storage> 0.3

According to the present invention, in a nucleic acid detection method using a probe, the decomposition of the probe in the nucleic acid detection reaction solution can be suppressed very easily and stably without changing conditions such as the detection procedure or performing a special pretreatment. Nucleic acid detection is possible.

In Example 1, it is the figure which showed the amplification curve of qPCR using a probe (IL6). In Example 1, it is the figure which confirmed the fluorescence intensity by Multicomponent of qPCR using a probe (IL6). In Example 1, it is the figure which showed the amplification curve of qPCR using a probe (IL6). In Example 1, it is the figure which confirmed the fluorescence intensity by Multicomponent of qPCR using a probe (IL6). In Example 2, it is the figure which showed the amplification curve of qPCR using a probe (CDK10). In Example 2, it is the figure which confirmed the fluorescence intensity by Multicomponent of qPCR using a probe (CDK10).

Hereinafter, the present invention will be described in detail.
One embodiment of the present invention is a nucleic acid detection method that uses a probe and allows a nucleic acid detection reaction solution to coexist with ammonium sulfate and / or tetramethylammonium acetate.

(probe)
In the present invention, a “probe” is an oligonucleotide having a base sequence selected by a target nucleic acid to be detected. The sequence and length of the probe are not particularly limited, and are appropriately determined by a conventionally known method depending on the target sequence.
The probe may bind an enzyme such as alkaline phosphatase, a labeling substance such as biotin, avidin, or a fluorescent dye. The label of the probe is not limited, but a fluorescent dye is preferable. The labeling position of the labeled probe is preferably labeled at or near the end in the base sequence.

(Nucleic acid detection method)
The “nucleic acid detection method” in the present invention is not particularly limited as long as it is a method for detecting a nucleic acid using a probe. It may be accompanied by nucleic acid amplification or may not be accompanied. Examples of methods that do not use nucleic acid amplification include Fluorescence in situ hybridization (FISH) method, Hybrid Capture method, and Invader method.
The nucleic acid detection method may involve a nucleic acid amplification step. In this case, amplification may be performed before detection or may be performed simultaneously with detection.
Although the nucleic acid detection method accompanying amplification is not specifically limited, For example, PCR method is mentioned. In addition, the method is not limited to this, for example, a Loop-Mediated Is Amplification (LAMP) method, a Transcribation Reverse Transformed Reaction Reaction (TRC) method, or a Nucleic Acid Sequencing Method (NBA).

When the nucleic acid detection method is qPCR, examples of the probe include TaqMan (registered trademark) Probe, Molecular Beacon, Hybridization Probe, Cycling Probe, and Q Probe, and any probe can be selected.

(Nucleic acid detection reaction solution)
The “nucleic acid detection reaction solution” in the present invention is not particularly limited as long as components (substances) necessary for carrying out the nucleic acid detection reaction are prepared. In general, it preferably contains a metal ion necessary for nucleic acid hybridization. When the nucleic acid detection method is qPCR, it preferably contains a substrate such as a primer, DNA polymerase, deoxynucleoside triphosphate, etc., and further contains a metal ion.

When the nucleic acid detection method involves amplification by DNA polymerase, the “DNA polymerase” used in the nucleic acid detection reaction solution is not particularly limited. DNA polymerases derived from various thermostable bacteria can be used. Specific examples include Taq DNA polymerase and Tth DNA polymerase belonging to family A (PolI type), KOD DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase belonging to family B (α type), and the like. Among them, Taq DNA polymerase and Tth DNA polymerase are preferable because they have 5 '→ 3' exonuclease activity and are used in the TaqMan (registered trademark) Probe method, but are not limited thereto.

(Method for determining ammonium sulfate and tetramethylammonium acetate)
In the present invention, when quantifying ammonium sulfate and / or tetramethylammonium acetate in a nucleic acid detection reaction solution, it is detected by the GC / MS method.

(Effect of the present invention)
By using the nucleic acid detection method of the present invention, in the nucleic acid detection method using a probe, ammonium sulfate and / or tetramethylammonium acetate are allowed to coexist in the nucleic acid detection reaction solution, thereby suppressing degradation of the probe in the nucleic acid detection reaction solution. be able to.

(Probe disassembly)
In the present invention, the degree of “probe decomposition” can be quantitatively confirmed by the following method (1) or (2).
(1) The Ct value in PCR is compared between the probe that has been added to the reaction solution and the time elapsed and the sample that has just been added to the reaction solution.
(2) The fluorescence intensity at the beginning of the PCR cycle is compared between the probe that has been added to the reaction solution and the time has elapsed and the probe that has just been added to the reaction solution.

In the present invention, “suppressing the decomposition of the probe” ideally means adding the probe that decomposes by leaving it under a temperature condition of 5 to 35 ° C. to the reaction solution, and before and after storing it at room temperature. When measured by the method, it means that the Ct value is equivalent after storage or the fluorescence intensity at the beginning of the cycle is equivalent after storage.
Specifically, TaqMan (registered trademark) Gene Expression Assays of Applied Biosystems, Gene Name: Interleukin 6, Assay ID: Hs00985639_m1 (hereinafter abbreviated as “IL6”), or Gene Name: cyclin-dependent ID, When the nucleic acid detection reaction solution to which Hs00177586_m1 (hereinafter abbreviated as “CDK10”) is added is allowed to stand at room temperature for 24 hours and the following equation is satisfied, the probe decomposition is suppressed.
(1) Ct value before storage / Ct value after storage> 0.8
(2) Fluorescence intensity at the beginning of the cycle before storage / Fluorescence intensity at the beginning of the cycle after storage> 0.3
The value in (1) is more preferably 0.9 or more. The value in (2) is more preferably 0.5 or more.

In this specification, after the nucleic acid detection reaction solution is stored at 25 ° C. for 3 hours or longer, the Ct value or the fluorescence intensity at the beginning of the cycle is compared before and after storage based on the evaluation method, and at least one of them is compared. Are equal, it is concluded that probe degradation is suppressed, and it is determined that the probe is stabilized.
If the degradation of the probe was suppressed after the test was further extended at 25 ° C. (64 hours), the degree of stabilization was judged to be higher depending on the length of the storage time. it can.

As described above, the present inventors have examined the PCR method as an example, and found that the degradation of the probe in the nucleic acid detection reaction solution is due to the 5 ′ → 3 ′ exonuclease activity of the DNA polymerase.
The enzyme having the nuclease activity is not limited to the DNA polymerase having the 5 ′ → 3 ′ exonuclease activity described above. Examples thereof include DNase I, DNase II, DNA polymerase, and restriction enzyme, which are generally said to require metal ions such as magnesium, calcium, manganese, and zinc.
These enzymes may be derived from those used for nucleic acid amplification and contained in the nucleic acid detection reaction solution, such as DNA polymerase in the PCR method, or derived from contaminants that should not normally be included in the nucleic acid detection reaction solution. You may do it.

(Method of allowing ammonium sulfate and / or tetramethylammonium acetate to coexist in the nucleic acid detection reaction solution)
In the present invention, the method for allowing ammonium sulfate or tetramethylammonium acetate to coexist in the nucleic acid detection reaction solution is not particularly limited. Only either ammonium sulfate or tetramethylammonium acetate may coexist, or both may coexist.

Ammonium sulfate and / or tetramethylammonium acetate may be added in advance to the nucleic acid detection reaction solution, or may be added to the reaction solution together with the probe. However, when the nucleic acid detection reaction solution contains an enzyme necessary for the detection reaction (for example, DNA polymerase), ammonium sulfate and / or tetramethylammonium acetate is added to the reaction solution at the same time as the probe or before the probe. It is preferable.
In addition, when the nucleic acid detection reaction solution is divided into several compositions before starting the reaction, for example, in a nucleic acid detection kit, etc., and is provided to be mixed and used during the reaction, the solution is divided. It may be added to any one or more of the compositions.

  In the present invention, the concentration of ammonium sulfate and / or tetramethylammonium acetate in the nucleic acid detection reaction solution is preferably 1 mM to 40 mM, more preferably 1 mM to 20 mM, and even more preferably 1 mM to 10 mM.

  According to the present invention, it becomes possible to easily and effectively suppress the decomposition of the probe, and a remarkable effect is exhibited as compared with the prior art.

Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not particularly limited by the examples.

Example 1: Probe decomposition and stabilization effect by adding ammonium sulfate and / or tetramethylammonium acetate qPCR for the purpose of confirming probe decomposition by standing at room temperature and probe stabilization by ammonium sulfate and tetramethylammonium acetate The analysis was performed using.
As a sample, cDNA prepared from HeLa cell (cell derived from human cervical cancer) RNA was used. For extraction of RNA and preparation of cDNA, SuperPrep (registered trademark) Cell Lysis & RT Kit for qPCR (Toyobo Co., Ltd.) using reverse transcriptase derived from MMLV was used, and the procedure was in accordance with the instruction manual. 20 μL of each reaction solution was prepared using THUNDERBIRD (registered trademark) Probe qPCR Mix (Toyobo Co., Ltd.). The TaqMan probe and primer used were TaqMan (registered trademark) Gene Expression Assays, Gene Name: Interleukin 6, Applied ID: Hs00985639_m1 (hereinafter abbreviated as “IL6”) of Applied Biosystems. This product is a 20 times concentrated primer / probe mixture. The reaction was performed using a real-time PCR apparatus (Applied Biosystems 7500 Fast real-time PCR system) with a schedule of repeating 95 cycles of 95 ° C, 15 seconds → 60 ° C, 60 seconds, after a previous reaction at 95 ° C for 1 minute.
The concentration of the probe in the prepared reaction solution is 0.25 μM.

The prepared reaction solution was allowed to stand at 25 ° C. for 3 hours.
(Effect of ammonium sulfate)
An amplification curve obtained by qPCR using the probe (IL6) is shown in FIG. The numbers in the figure are as follows.
1: Probe was added after leaving the reaction solution at room temperature (Ct 31.7, 31.8) 2: 10 mM ammonium sulfate added (Ct 32.5, 32.7) 3: No addition (Ct 33.2, 33.9)

As a result, when the probe IL6 was not added, a delay in the Ct value was observed as compared with the control in which the probe was added for 3 hours (FIG. 1, No. 1, 3). On the other hand, when ammonium sulfate was added, a Ct value closer to the control to which the probe was added after 3 hours was shown compared to the case where ammonium sulfate was not added (FIG. 1, Nos. 1 to 3).

FIG. 2 shows the result of confirming the fluorescence intensity with multicomponent qPCR using the probe (IL6). The numbers in the figure are as follows.
1: Add probe after leaving the reaction solution at room temperature 2: Add 10 mM ammonium sulfate 3: No addition

At this time, the fluorescence intensity at the beginning of the cycle is high when not added (FIG. 2; No. 3). This is presumably because the probe was decomposed before starting the cycle due to standing at room temperature, the fluorescent label was released from the probe, the quenching by the quencher was released, and fluorescence was emitted. On the other hand, in the control to which ammonium sulfate was added or in which the probe was added after 3 hours, the fluorescence intensity at the beginning of the cycle was low (FIG. 2; No. 1 and 2). This is probably because the probe is not decomposed and quenching by the quencher is maintained.

(Effect of tetramethylammonium acetate)
FIG. 3 shows an amplification curve of qPCR using the probe (IL6). The numbers in the figure are as shown below.
1: Probe was added after leaving the reaction solution at room temperature (Ct 31.7, 31.8) 2: 20 mM tetramethylammonium acetate added (Ct 32.1, 32.2) 3: No addition (Ct 33.2, 33.9)
FIG. 4 shows a graph in which the fluorescence intensity is confirmed by a multicomponent of qPCR when the probe (IL6) is used. The reaction solution was left at room temperature for 3 hours after preparation. 20 mM tetramethylammonium acetate was added. The numbers in the figure are as follows.
1: Add probe after leaving the reaction solution at room temperature 2: Add 20 mM tetramethylammonium acetate 3: No addition

Regarding tetramethylammonium acetate, a tendency similar to that of ammonium sulfate was observed, and when 20 mM tetramethylammonium acetate was added, an improvement in Ct value delay was observed (FIG. 3; No). .2, 3).
At this time, the fluorescence intensity at the beginning of the cycle can be suppressed to a low level by adding tetramethylammonium acetate, as in ammonium sulfate (FIG. 4).
However, even when ammonium sulfate or tetramethylammonium acetate is added, it is not as low as the control in which the probe was added after 3 hours. Therefore, the decomposition of the probe is not completely suppressed, and it is considered that the addition concentration adjustment is necessary to obtain a more preferable effect (FIGS. 2 and 4).

Example 2: Unstable probe stabilization effect due to addition of ammonium sulfate For the purpose of confirming in detail the effect of inhibiting decomposition of the probe by standing at room temperature, the concentration of ammonium sulfate was changed using an unstable probe different from Example 1. It added by changing and it analyzed using qPCR.
qPCR followed the method of Example 1. TaqMan (registered trademark) Gene Expression Assays, Gene Name: cyclin-dependent kinase 10 and Assay ID: Hs00177586_m1 (hereinafter abbreviated as “CDK10”) of Applied Biosystems were used as TaqMan probes and primers.
The prepared reaction solution was allowed to stand at 25 ° C. for 64 hours.

The amplification curve obtained by qPCR is shown in FIG. The numbers in the figure are as shown below.
1: Add probe after leaving the reaction solution at room temperature (Ct29.2, 28.7) 2: Add 40 mM ammonium sulfate (Ct29.2, 29.3) 3: Add 20 mM ammonium sulfate (Ct30.5, undetectable) 4: Addition of 10 mM ammonium sulfate (Ct35.8, 37.3) 5: No addition (not detectable, not detectable)

As a result, no Ct value was detected in the probe CDK10 when it was not added (FIG. 5, No. 5). On the other hand, when ammonium sulfate was added, a Ct value close to the control in which the probe was added after 64 hours was shown in a concentration-dependent manner (FIG. 5, Nos. 1 to 4).

FIG. 6 shows the result of confirming the fluorescence intensity with multicomponent of qPCR using the probe (CDK10). The numbers in the figure are as follows.
1: Add probe after standing at room temperature 2: Add 40 mM ammonium sulfate 3: Add 20 mM ammonium sulfate 4: Add 10 mM ammonium sulfate 5: No addition

At this time, the fluorescence intensity at the beginning of the cycle is highest when not added, and as the concentration of ammonium sulfate increases, it is closer to the control to which the probe was added after 64 hours (FIG. 6). This is considered to be because the probe is not decomposed and the quenching by the quencher is maintained as the concentration of ammonium sulfate is increased.

The present invention works effectively to stabilize the probe. This enables stable data collection when analyzing a large amount of gene expression in which the reaction solution is assumed to be left at room temperature. The present invention is particularly useful when gene expression analysis is performed on a large amount of specimens in which a prepared reaction solution is assumed to be left at room temperature for a long time. It can also be used for inspection.

Claims (4)

A method for stabilizing a probe, characterized in that ammonium sulfate and / or tetramethylammonium acetate coexist in a nucleic acid detection reaction solution used in a nucleic acid detection method using a probe. The method for stabilizing a probe according to claim 1, wherein ammonium sulfate and / or tetramethylammonium acetate are allowed to coexist in the nucleic acid detection reaction solution at a concentration of 1 mM to 40 mM. The method for stabilizing a probe according to claim 1 or 2, wherein the nucleic acid detection method is PCR using a probe. The nucleic acid detection solution is stored for 24 hours under a temperature condition of 5 ° C to 35 ° C, and then satisfies the requirement of either (a) or (b). To stabilize the probe.
(A) Ct value before storage / Ct value after storage> 0.8
(B) Fluorescence intensity at the beginning of the cycle before storage / Fluorescence intensity at the beginning of the cycle after storage> 0.3