JP6834164B2 - How to maintain the detection sensitivity of PCR reaction solution - Google Patents

Description

The present invention relates to the field of nucleic acid amplification. More specifically, the present invention relates to a method of extending the storage time while maintaining the detection sensitivity of the target nucleic acid by suppressing the decomposition of the oligonucleotide primer (primer) in the PCR reaction solution.

The synthesis of DNA from a template nucleic acid using DNA polymerase is used and applied to various methods such as sequencing method and nucleic acid amplification method in the field of molecular biology. Among them, the nucleic acid amplification method has already been put into practical use not only in the research field but also in the forensic field such as genetic diagnosis and paternity testing, or in the microbiological examination in foods and the environment.

Among them, the PCR method used for amplifying a specific sequence of DNA is an extremely widespread technique from research fields to application fields. Currently, the PCR method is being further developed, and there are various technologies such as the Multiplex PCR method that simultaneously amplifies multiple primers and the real-time PCR method that detects the amplification product of PCR in real time using a fluorescent dye. To do. These technologies have already been put into practical use not only in research fields but also in forensic fields, foods, and microbial tests in the environment.

The gene amplification method by the PCR method is performed by repeating a cycle consisting of denaturation, annealing, and elongation in the presence of a target nucleic acid, four types of deoxynucleoside triphosphates, at least a pair of primer sets, and a DNA polymerase for 20 to 50 cycles. This is a method of exponentially amplifying the region of the target nucleic acid sandwiched between the pair of primer sets.

The PCR method has the characteristic that the target gene sequence can be amplified by amplifying the region of the target nucleic acid sandwiched between the pair of primers, but when the primer hybridizes to an incorrect sequence that is not the target. (Primer mismatch annealing) also has a drawback that unintended sequence amplification occurs and non-specific amplification occurs. In addition, the association of primers with each other during PCR causes a primer dimer, which acts as a template DNA to produce a non-specific amplification product. Therefore, the problem in PCR is to amplify only the target product without non-specific amplification.

A typical method for reducing non-specific amplification is a hot start method using an antibody. The hot start method is a method of reducing non-specific amplification caused by pre-cycle mismatch annealing and primer dimer by inhibiting the polymerase activity and exonuclease activity of DNA polymerase with a neutralizing antibody. However, such a method is often not sufficiently effective in suppressing non-specific amplification, or even suppresses specific amplification.

Furthermore, when the primer, DNA polymerase, and magnesium ion are contained in the same solution, degradation of the primer occurs due to the exonuclease activity of the DNA polymerase. In such a case, the specificity of the primer is lowered, non-specific amplification due to the primer dimer occurs, and as a result, the detection sensitivity of the target nucleic acid is lowered. Therefore, in order to suppress the exonuclease activity of DNA polymerase, it is necessary to always prepare the PCR reaction solution on ice, and there is a problem that the PCR reaction must be carried out promptly after the preparation of the PCR reaction solution. .. From the field of clinical examination, there is a strong need to solve these problems and allow the PCR reaction solution to stand at room temperature for a certain period of time.

As described above, it is possible to prepare a PCR reaction solution containing a primer, DNA polymerase and magnesium ion at room temperature (in the present invention, it means a temperature from 20 ° C. to 30 ° C.), and the prepared PCR reaction. There has been a demand for a method for sensitively detecting a target nucleic acid even after the solution has been allowed to stand at room temperature for a certain period of time or longer.

Japanese Patent No. 5830286

Against the background of the above circumstances, a PCR reaction solution can be prepared at room temperature by suppressing the decomposition of the primer with a solution containing the primer, DNA polymerase and magnesium ion, and further, the prepared PCR reaction solution can be prepared. An object of the present invention is to provide a method for detecting a target nucleic acid with high specificity even after standing at room temperature for a certain period of time or longer.

In view of the above circumstances, as a result of diligent research, the present inventors can suppress the generation of non-specific increasing width products by using a DNA polymerase having no exonuclease activity, and further, the DNA polymerase can be used. We have found that by using it, the formation of a primer dimer can be suppressed even in the coexistence of magnesium ion and a primer, and have completed the present invention.

That is, the present invention has the following configuration.
Item 1. A method for detecting by amplifying two or more types of target nucleic acids, which comprises the following steps (a) to (d).
(A) A step of preparing a PCR reaction solution containing two or more kinds of oligonucleotide primer sets for amplifying a target region in a nucleic acid, a DNA polymerase having no exonuclease activity, and magnesium ions.
(B) Step of mixing the sample with the PCR reaction solution,
(C) Amplification by the PCR reaction and
(D) Step 2. Detecting a signal indicating an amplification product in the target region. Item 2. The method according to Item 1, wherein the PCR reaction solution of (a) contains magnesium ions of 2 mM or more.
Item 3. Item 2. The method according to Item 1 or 2, wherein the PCR reaction solution according to (a) further contains an intercalator.
Item 4. Item 2. The method according to any one of Items 1 to 3, wherein the step of preparing the PCR reaction solution of (a) is carried out under a temperature condition of 20 ° C. to 30 ° C.
Item 5. The PCR reaction solution of (a) after preparation is allowed to stand for 4 hours under a temperature condition of 20 ° C. to 30 ° C., and is derived from the target nucleic acid obtained when the steps (b) to (d) are carried out. The signal is 30% or more as compared with the signal derived from the target nucleic acid obtained when the PCR reaction solution within 10 minutes after the preparation of (a) is used and the steps (b) to (d) are carried out. Item 4. The method according to any one of Items 1 to 4, wherein the method remains.
Item 6. A nucleic acid amplification kit for amplifying and detecting a target nucleic acid contained in a sample, at least two kinds of oligonucleotide primer sets for amplifying a target region of the nucleic acid, and a DNA polymerase having no exonuclease activity. And a kit for nucleic acid amplification, which comprises magnesium ions in the same solution.

According to the method of the present invention, a PCR reaction solution can be prepared at 20 ° C. to 30 ° C., and further, non-specific amplification even after the prepared PCR reaction solution is stored at 20 ° C. to 30 ° C. for a certain period of time or longer. No product is generated, and the target nucleic acid can be detected with high specificity.

This is the result of detecting the target nucleic acid using a PCR reaction solution using a DNA polymerase having exonuclease activity within 10 minutes and a solution left at 25 ° C. for 4 hours. This is the result of detecting the target nucleic acid using a PCR reaction solution using a DNA polymerase having no exonuclease activity within 10 minutes and a solution left at 25 ° C. for 4 hours. This is the detection result of the target nucleic acid when magnesium ion was added immediately before the start of the reaction.

Hereinafter, the present invention will be described in detail.
In the embodiment of the present invention, the PCR reaction solution contains an oligonucleotide primer set (primer set), a DNA polymerase having no exonuclease activity, and magnesium ions. Other configurations are not particularly limited, but contain a template nucleic acid, one or more kinds of deoxynucleotide triphosphates (dATP, dCTP, dTTP, dGTP), derivatives of deoxynucleotide triphosphates, buffers, salts, and the like. It is preferable to do so.

Here, as the buffer, for example, Tris (TRIS), Tricine (TRICINE), Bisutricin (BIS-TRICINE), HEPES, MOPS (MOPS), TES (TES), Taps (TAPS), Pipes (PIPES), And caps (CAPS) and the like, but are not particularly limited.

Examples of the reaction buffer include Tris-hydrochloric acid buffer, Tris-sulfate buffer, and tricine buffer. The concentration of the buffer solution is preferably about 10 mM to 200 mM, more preferably about 20 mM to 100 mM. The pH is preferably in the range of 7.0 to 9.5, more preferably in the range of 7.5 to 9.0.

The reaction composition of the present invention preferably contains 1 mM to 10 mM magnesium ions. More preferably it contains 2 mM to 7 mM, more preferably 3 mM to 5 mM magnesium ions. The form in which magnesium ions are added is not particularly limited, but it is preferable to use magnesium salts such as magnesium sulfate and magnesium chloride.

The primers used in the present invention are not particularly limited as long as they are designed to amplify the region of interest. In the present invention, in order to detect a plurality of DNA regions simultaneously and rapidly, two or more kinds, three or more kinds, or four or more kinds of primer sets are included, and each primer amplifies the target DNA. Further, either the forward side or the reverse side primer can be shared. The PCR reaction solution may further contain an internal standard gene and primers for amplification thereof for the purpose of confirming whether the PCR reaction is proceeding.

The heat-resistant DNA polymerase used in the present invention is not particularly limited as long as it is a DNA polymerase that does not exhibit exonuclease activity. 3'→ 5'exonuclease activity and 5'→ 3'exonuclease activity are known as exonuclease activities, but in the embodiment of the present invention, 3'→ 5'exonuclease activity and 5'→ 3'exonuclease are known. Use a DNA polymerase that does not show any nuclease activity. The DNA polymerase is not particularly limited as long as it does not show exonuclease activity, and wild-type DNA polymerase that does not show exonuclease activity, DNA polymerase that lacks exonuclease activity by mutation, and the like can be used.

The DNA polymerase used in the present invention in which the exonuclease activity is deleted by mutation is not particularly limited, and examples thereof include the mutant Taq DNA polymerase disclosed in Patent Document 1. The Taq DNA polymerase is a modified Taq DNA polymerase in which the amino acid on the N-terminal side is deleted in order to reduce or delete the 5'→ 3'exonuclease activity.
The DNA polymerase used in the present invention is not particularly limited as long as it is a DNA polymerase that does not exhibit exonuclease activity, and its origin, production method, and mutation site are not particularly limited.

The detection means in the step of detecting the signal indicating the amplification product of the target region in the present invention is not particularly limited, and examples thereof include melting curve analysis and electrophoresis. Especially, melting curve analysis is preferable.

In the present invention, as a method for detecting an amplification product using a fluorescent substance by melting curve analysis, an intercalator to which a fluorescent dye that binds to double-stranded DNA is added using an intercalator such as SYBR (registered trademark) Green. The law can be mentioned. An intercalator is any molecule that binds to a nucleic acid in a reversible, non-covalent manner by inserting (intercalating) into double-stranded DNA, thereby indicating the presence and amount of nucleic acid. .. In general, an intercalator is a dye that is inserted into double-stranded DNA and emits fluorescence.

In the present invention, it is possible to use many types of intercalators. For example, Ethidium bromede, cyanine dyes (eg, TOTO®, YOYO®, BOBO and POPO), SYBR® Green I, SYBR® Green ER, SYBR® Green Gold. , SYBR® DX, PicoGreen®, LCGeen®, EvaGreen®, SYSTEMX® Green, ResoLight, Propidium iodide, Acidine orange, 7-amino-actinomycin D, CyQUANT (registered trademark) GR, SYTO (registered trademark) 9, SYTO (registered trademark) 10, SYTO (registered trademark) 13, SYTO (registered trademark) 14, SYTO (registered trademark) 82, FUN-1 and the like. , Not particularly limited.

The signal derived from the target nucleic acid in the present invention refers to a negative value (-dF / dT) of the first derivative of fluorescence derived from the intercalator in the melting curve analysis. Further, when the peak (Tm value) of the differential value is in the temperature range within 1 ° C. before and after, it is determined that the signal is derived from the same sample.

In the present invention, when comparing signals derived from the same sample, a PCR reaction solution that has been allowed to stand at 25 ° C. for 4 hours is used as compared with the signal detected using the PCR reaction solution within 10 minutes after preparation. The negative value (−dF / dT) of the first derivative value of the detected fluorescence at the same Tm value remains 30% or more, preferably 40% or more, more preferably 50%.

According to the present invention, even when the mixture is allowed to stand at 25 ° C. for 4 hours, the decrease in signal can be suppressed, so that the reagent once prepared can be used as it is, and as a result, clinical tests and the like can be used. It will be possible to significantly improve workability in the field. Needless to say, it can be used for a longer time than 4 hours by allowing it to stand under refrigerated conditions instead of at room temperature.

In addition to the above, the reaction solution used in the nucleic acid amplification method of the present invention may contain uracil glycosidase and dUTP as a composition for preventing carryover contamination. Uracil glycosidase is an enzyme that specifically excises uracil present in a DNA sequence and causes DNA degradation. The DNA fragment obtained by PCR with a composition containing dUTP in the reaction solution contains uracil and is degraded by uracil glycosidase. On the other hand, natural DNA does not contain uracil and is not degraded. A method of preventing carryover contamination using this is known.

The kit of the present invention is a kit for use in the detection method of the present invention. That is, the kit of the present invention contains a sample containing two or more, three or more, or four or more primer sets for amplifying a target region in a nucleic acid, a DNA polymerase having no exonuclease activity, and magnesium ions. It is a kit for amplifying the nucleic acid contained in and detecting it by melting curve analysis.

The primers, DNA polymerase and magnesium ion used in the present invention are as described above. The kit of the present invention may also further include reagents required for nucleic acid amplification and melting curve analysis. Specifically, it may contain one or more kinds of deoxynucleotide triphosphates (dATP, dCTP, dTTP, dGTP) or derivatives, buffers, and salts of deoxynucleotide triphosphates, and the above-mentioned dUTP.
The kit is characterized by containing primers, DNA polymerase, and magnesium ions as a mixture, but other reagents may be contained separately or some of them may be mixed. Good.

Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not limited to the following examples.

Example 1. Effect of exonuclease activity of DNA polymerase on PCR reaction fluidity Multiplex PCR reaction using 10 copies each of salmonella gene, enterohemorrhagic Escherichia coli (EHEC), and diarrhea bacterium genomic DNA as samples And melting curve analysis was performed. The peak of the internal standard gene is given at 71 ° C, the peak of Salmonella is given at 76 ° C, the peak of enterohemorrhagic Escherichia coli is given at 80 ° C, and the peak of Shigella is given at 85 ° C.

In this example, as a primer set for multiplex PCR, a 10 × primer mix contained in an intestinal bacterial gene detection kit-fast fluorescence detection- (TOYOBO) was used. As the DNA polymerase of this example, rTaq DNA polymerase (TOYOBO) was used as a DNA polymerase having exonuclease activity. Other reaction solution compositions are shown in Table 1.

A PCR reaction solution prepared at 25 ° C. within 10 minutes and a PCR reaction solution prepared at 25 ° C. and allowed to stand at the same temperature for 4 hours were used. Ten copies of each purified genomic DNA were added to these PCR reaction solutions, and PCR reaction and melting curve analysis were performed. For melting curve analysis, PCR was performed under the cycle conditions shown in Table 2 to detect signals derived from the target nucleic acid. A real-time PCR device, Light Cycler® 96 (Roche), was used for the analysis.

As a result of the analysis, in the PCR reaction solution using DNA polymerase having exonuclease activity, compared with the PCR reaction solution within 10 minutes after preparation, in the PCR reaction solution left to stand for 4 hours after preparation, the part standard gene, Salmonella, The signal indicating the target gene of the diarrhea was lost. The signal indicating the target gene of enterohemorrhagic Escherichia coli remained, but the survival rate was about 30%.

Example 2. Effect of exonuclease activity of DNA polymerase on PCR reaction humorality Multiplex PCR reaction and melting curve analysis using 10 copies each of salmonella gene, enterohemorrhagic Escherichia coli (EHEC), and Shigella genomic DNA as samples Was carried out. The peak of the internal standard gene is given at 71 ° C, the peak of Salmonella is given at 76 ° C, the peak of enterohemorrhagic Escherichia coli is given at 80 ° C, and the peak of Shigella is given at 85 ° C.

In this example, Titanium Taq DNA Polymerase (CLONTECH LABORATORIE) was used as a DNA polymerase having no exonuclease activity. Other reaction solution compositions are shown in Table 1.

A PCR reaction solution prepared at 25 ° C. within 10 minutes and a PCR reaction solution prepared at 25 ° C. and allowed to stand at the same temperature for 4 hours were used. Ten copies of each purified genomic DNA were added to these PCR reaction solutions, and PCR reaction and melting curve analysis were performed. For melting curve analysis, PCR was performed under the cycle conditions shown in Table 2 to detect signals derived from the target nucleic acid. A real-time PCR device, Light Cycler® 96 (Roche), was used for the analysis.

As a result of the analysis, in the PCR reaction solution using DNA polymerase having no exonuclease activity, all the signals derived from the four target genes including the internal standard were observed as the signals of the internal standard. Compared with the PCR reaction solution within 10 minutes after preparation, in the PCR reaction solution left to stand for 4 hours after preparation, 100% of the signals indicating the internal standard gene, Salmonella, and Shigella remain, and the target of enterohemorrhagic Escherichia coli About 65% of the signal indicating the gene remained.

Example 3. Effect of Magnesium Ion on PCR Reaction Liquidity Multiplex PCR reaction and melting curve analysis were performed using 10 copies each of Salmonella gene, enterohemorrhagic Escherichia coli (EHEC), and Shigella genomic DNA as samples. The reaction solution shown in Table 3 was prepared at 25 ° C. and allowed to stand at 25 ° C. for 4 hours. As this reaction solution, rTaq DNA polymerase (TOYOBO) having exonuclease activity was used. Magnesium sulfate was added to the reaction solution after standing so that the final concentration was 3 mM. Ten copies of each purified genomic DNA were added to this PCR reaction solution, and PCR reaction and melting curve analysis were performed. For melting curve analysis, PCR was performed under the cycle conditions shown in Table 2 to detect signals derived from the target nucleic acid. A real-time PCR device, Light Cycler® 96 (Roche), was used for the analysis.

As a result of the analysis, signals derived from four target genes including the internal standard were observed as the signals of the internal standard. The survival rate was 90% or more for each of the four signals.

According to the present invention, by using a DNA polymerase having no exonuclease activity, it is possible to improve the stability at room temperature even in a PCR reaction solution composition containing magnesium ions and oligonucleotide primers in the same solution. .. In addition, conventionally, a kit containing a magnesium ion or an oligonucleotide primer in a separate container has been used in order to improve stability, but by using the method of the present invention, a DNA polymerase, a magnesium ion and an oligonucleotide primer can be obtained. It is possible to create a kit to be contained in the same solution. Therefore, since the preparation of reagents becomes simpler, it becomes possible to easily perform genetic testing using nucleic acid amplification detection even in clinical diagnosis, which is often performed by a person who does not have specialized knowledge in nucleic acid amplification technology. , Is expected to contribute significantly to the industrial world.

Claims (4)

A nucleic acid amplification kit for amplifying and detecting a target nucleic acid contained in a sample, at least two kinds of oligonucleotide primer sets for amplifying a target region of the nucleic acid, and a DNA polymerase having no exonuclease activity. A kit for nucleic acid amplification, which comprises the same solution containing magnesium ions in the same solution and capable of specifically detecting the target nucleic acid even after storage at 25 ° C. for 4 hours as a PCR reaction solution. The nucleic acid amplification kit according to claim 1 , wherein the PCR reaction solution contains magnesium ions of 2 mM or more. The nucleic acid amplification kit according to claim 1 or 2 , wherein the PCR reaction solution further contains an intercalator. The PCR reaction solution is allowed to stand for 4 hours under a temperature condition of 20 ° C. to 30 ° C., and then (b) a step of mixing a sample, (c) a step of amplifying by a PCR reaction, and (d) a step of a target region. The signal derived from the target nucleic acid obtained when the step of detecting the signal indicating the amplification product is obtained by using the PCR reaction solution within 10 minutes after the preparation of the PCR reaction solution according to the above (b) to (d). The nucleic acid amplification kit according to any one of claims 1 to 3 , which makes it possible to retain 30% or more of the signal derived from the target nucleic acid obtained when the step is carried out.