JP2018033324A - Method of detecting target nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of detecting a marker probe comprising an oligonucleotide modified with a fluorescent substance at one terminal and a quencher substance at the other terminal without requiring a purification step of a biological sample.SOLUTION: The method detects the presence of target nucleic acid from a biological sample without purifying the nucleic acid by isolating labelled fragments cleaved from marker probes hybridized to the target nucleic acid using (a) DNA polymerase with no 5'-3' exonuclease activity and (b) Flap endonuclease (FEN) and detecting or measuring the isolated labelled fragments.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting or measuring a target nucleic acid sequence in nucleic acid amplification, particularly PCR (Polymerase Chain Reaction).

The PCR method is (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) primer annealing to template single-stranded DNA, and (3) the above using DNA polymerase This is a method of amplifying a target nucleic acid in a sample by repeating three cycles of three steps of primer extension as one cycle. This method makes it possible to amplify a target nucleic acid several hundred thousand times from a trace amount sample such as several copies, which is an indispensable technique for genetic research.

In particular, real-time PCR can easily detect and quantify a small amount of nucleic acid without performing electrophoresis after the reaction, so it can be used not only for research purposes but also in forensic fields such as genetic diagnosis and clinical diagnosis, or in food and the environment. Also widely used in microbial testing and the like. Amplification using PCR reaches a certain intensity in a short time when the copy number of the nucleic acid as a template is large, and takes a long time when the copy number is small. For this reason, in real-time PCR that can measure the time course of amplification using fluorescence, the template nucleic acid can be quantified by comparing the number of amplifications reaching a certain intensity with a nucleic acid of known concentration and a nucleic acid of unknown concentration.

Real-time PCR fluorescence detection methods include an intercalator method using a fluorescent dye that binds to double-stranded DNA, and a hybridization method that uses a fluorescent probe that specifically binds to a gene product. As an example of the former, a fluorescent dye called SYBR (registered trademark) Green is widely used. SYBR (registered trademark) Green is highly versatile because fluorescent dyes that bind to double-stranded DNA can be used for any sequence, but it also binds to non-specific amplification products such as primer dimerization. Inaccurate measurements may be obtained. In contrast, hybridization methods represented by TaqMan (registered trademark) probes and FRET probes require the preparation of fluorescent probes having specific sequences, depending on the gene product. There are advantages you can do.

In diagnostic applications, it is important that there is no detection of non-specific amplification products, and it is important to be able to analyze with a multiplex by changing the fluorescent dye, and detection by the hybridization method is common. The TaqMan (registered trademark) probe is most widely used because it is easy to design, and various diagnostic agents using the TaqMan (registered trademark) probe have been developed. A TaqMan (registered trademark) probe is a probe in which a fluorescent dye is labeled at the 5 ′ end of a target nucleic acid-specific oligonucleotide and a quencher is labeled at the 3 ′ end, and is hydrolyzed by the 5′-3 ′ exonuclease activity of the PCR enzyme. Thus, a fluorescent signal is emitted, and detection of the fluorescent signal enables the detection and quantification of a small amount of nucleic acid.

The thermostable DNA polymerase used for PCR includes those belonging to Family A and those belonging to Family B. Known DNA polymerases belonging to family A include DNA polymerase (Tth DNA polymerase) derived from Thermus thermophilus and DNA polymerase (Taq DNA polymerase) derived from Thermus aquaticus. Yes. In addition, DNA polymerases belonging to Family B include thermostable DNA polymerase (Pfu DNA polymerase) derived from Pyrococcus furiosus and thermostable DNA polymerase (Tli DNA polymerase) derived from Thermococcus litoralis. ), Thermostable DNA polymerase (KOD DNA polymerase) derived from Thermococcus kodakaraensis, and the like are known.

Recently, DNA polymerases belonging to family B have been evaluated in that they do not depend on the sequence of the amplification target and are strong against PCR inhibition, and there is a trend to use DNA polymerases belonging to family B for diagnostic purposes. However, DNA polymerase belonging to family A uses its own 5′-3 ′ exonuclease activity and can detect TaqMan probes, while DNA polymerase belonging to family B has 5′-3 ′ exonuclease activity. Therefore, there is a problem that it cannot be used for detecting a TaqMan (registered trademark) probe.

In addition, DNA polymerase belonging to Family A tends to depend on the target sequence, and it is difficult to amplify GC-rich or AT-rich targets. It has been reported that DNA polymerase belonging to Family A can be amplified by removing exonuclease activity (Patent Document 2), but detection with a TaqMan (registered trademark) probe is similar to the above. There was a problem that could not be.

As a conventionally reported technique, there is known a method for detecting the presence of a target nucleic acid by promoting cleavage of FEN nuclease by inserting a mismatch at the 5 'end of a labeled probe (Patent Document 3). However, in this method, it is necessary to insert a mismatched sequence into the probe sequence, which may cause non-specific detection.

Also known is a method of detecting a labeled probe in which a 5 'region is complementary to a target nucleic acid using a protein obtained by fusing FEN and a DNA polymerase belonging to family B (Patent Document 4). However, in this method, the stability of the fusion protein may be deteriorated, and it has been suggested that the reaction efficiency of DNA polymerase and the cleavage efficiency by FEN are reduced by using the fusion protein.

On the other hand, in diagnostic applications, detection from microorganisms having sequences biased to GC or AT, such as filamentous fungi and malaria, is necessary, and TaqMan (registered trademark) probes can be detected and PCR reactions that do not depend on the sequence of the amplification target A system was sought.

Japanese Patent Publication No. 6-500021 US Pat. No. 5,436,149 Japanese Patent No. 4960560 International Publication 2015/085230 Pamphlet

Currently, it is a family A that can be detected using a labeled probe composed of an oligonucleotide modified with a fluorescent substance at one end and a quencher substance at the other end, represented by TaqMan (registered trademark) probes. The DNA polymerase to which it belongs tends to depend on the sequence of the amplification target, and it was difficult to amplify GC-rich or AT-rich targets. There has been a demand for a method capable of detecting a labeled probe made of an oligonucleotide having a fluorescent substance modified at one end and a quencher substance modified at the other end without depending on the sequence of the amplification target.

As a result of intensive studies in view of the above circumstances, the present inventors have added FEN nuclease to a DNA polymerase belonging to family B that is resistant to PCR inhibitors and a DNA polymerase belonging to family A that lacks exonuclease. The present inventors have found that GC-rich and AT-rich targets can be detected with a TaqMan (registered trademark) probe, and have completed the present invention.

The representative present invention is as follows.
Item 1. A method for detecting a target nucleic acid having a GC rate or AT rate of 30% or less or 70% or more,
(A) a DNA polymerase having no 5′-3 ′ exonuclease activity, and
(B) Flap endonuclease (FEN)
A method for detecting the presence of a target nucleic acid by cleaving a labeled probe that hybridizes to a target nucleic acid to release a labeled fragment, and detecting or measuring the released labeled fragment.
Item 2. Item 2. The method according to Item 1, wherein the labeled probe is an oligonucleotide modified with a fluorescent substance at the 5 ′ end and a quencher substance at the 3 ′ end.
Item 3. The DNA polymerase having no 5′-3 ′ exonuclease activity is
(A) a DNA polymerase derived from family A that lacks 5′-3 ′ exonuclease activity, or
(B) selected from any of the DNA polymerases belonging to Family B, and
Item 3. The method according to Item 1 or 2, wherein the process is a DNA polymerase having 50 bases or more.
Item 4. The DNA polymerase having no 5′-3 ′ exonuclease activity is
(A) a DNA polymerase derived from family A that lacks 5′-3 ′ exonuclease activity, or
(B) selected from any of the DNA polymerases belonging to Family B, and
Item 4. The method according to any one of Items 1 to 3, which is a DNA polymerase having strand displacement ability.
Item 5. Item 5. The method according to any one of Items 1 to 4, wherein the FEN nuclease is a FEN nuclease whose activity is maintained at 50% or more even when heated at 60 ° C or more for 10 minutes.
Item 6. Item 7. The method according to any one of Items 1 to 6, further comprising an unpurified biological sample in the reaction solution.
Item 7. Item 7. The method according to Item 6, wherein the biological sample is any one selected from the group consisting of blood, feces, saliva, and urine.
Item 8. Item 8. A reagent for performing a method for detecting the presence of a target nucleic acid according to any one of Items 1 to 7, wherein the following (a) and (b) are contained in a reaction solution.
(A) DNA polymerase having no 5′-3 ′ exonuclease activity (b) Flap endonuclease (FEN)
Item 9. A kit for performing a method for detecting the presence of a target nucleic acid containing the reagent according to Item 8.

According to the present invention, it is possible to provide a PCR reaction solution that can be detected with a TaqMan (registered trademark) probe without depending on the sequence of the amplification target. In particular, it is very useful in diagnostic applications.

Comparison of detection with probe with and without 5 'terminal mismatch Evaluation of FEN-KOD fusion protein Comparison of detection sensitivity Amplification comparison of GC-rich targets Amplification detection from blood

Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.

The method for detecting the presence of the target nucleic acid in the present invention comprises:
(A) a DNA polymerase having no 5′-3 ′ exonuclease activity, and
(B) Flap endonuclease (FEN)
Is used to cleave the labeled probe that hybridizes to the target nucleic acid to release the labeled fragment, and detect or measure the released labeled fragment to detect the presence of the target nucleic acid.

The GC ratio of the target nucleic acid in the present invention indicates the ratio of G and C in the target nucleic acid sequence composed of A, T, G, C, and other modified bases. The ratio of G and C may be 30% or less of the whole or 70% or more, and the arrangement is not particularly limited.

The DNA polymerase having no 5'-3 'exonuclease activity in the present invention is not particularly limited as long as it is a DNA polymerase not having 5'-3' exonuclease activity. Preferably, as described in Patent Document 2, a deletion or mutation inserted into a polymerase belonging to family A to remove exonuclease activity, or DNA originally belonging to family B that does not have 5′-3 ′ exonuclease activity Examples include polymerases.

A DNA polymerase belonging to Family A from which 5′-3 ′ exonuclease activity has been removed includes a complete lack of 5′-3 ′ exonuclease activity, eg, 0.03%, 0. Refers to a modified DNA polymerase having 5′-3 ′ exonuclease activity of 05%, 0.1%, 1%, 5%, 10%, 20%, or at most 30%. A method for analyzing the activity of 5'-3 'exonuclease is described in Proc. Natl. Acad. Sci. USA Vol. 65, p168-175 (1970).

Examples of the DNA polymerase belonging to Family B include DNA polymerase (KOD) derived from Thermococcus kodakaraensis, DNA polymerase (Pfu) derived from Pyrococcus furiosus, and DNA polymerase (TGO derived from Thermococcus gorgonarius). ), DNA polymerase derived from Thermococcus litoralis (Tli, Vent), DNA polymerase derived from Pyrococcus sp. GB-D, DNA polymerase derived from Thermococcus sp. JDF-3, Thermococcus sp. 9 ° N DNA polymerase derived from -7, DNA polymerase derived from Thermococcus sp KS-1, DNA polymerase derived from Thermococcus cellar, or Thermococcus sicus A DNA polymerase derived from include, but are not particularly limited.

The DNA polymerase of the present invention may be any DNA polymerase that does not have 5′-3 ′ exonuclease activity, and further has a deletion or mutation introduced therein, or a fusion of SSB (single-stranded DNA binding domain). May be. The type of mutation is not particularly limited, and any DNA polymerase belonging to family A includes, for example, mutations described in Japanese Patent No. 580959 and Japanese Patent No. 04193997. If it is DNA polymerase which belongs to the family B, the variation | mutation as described in Unexamined-Japanese-Patent No. 2014-079236, Unexamined-Japanese-Patent No. 2014-079235, Unexamined-Japanese-Patent No. 2014-079233, patent 3891330 etc. is mentioned.

More preferably, it is desirable to use a DNA polymerase having a processivity of 50 bases or more. Preferably, in a DNA polymerase belonging to Family A, KlenTaq indicating 50 to 85 bases of processivity, and in a DNA polymerase belonging to Family B, , KOD, Phusion and the like showing processability of 300 bases or more. Here, the processivity indicates the number of bases that are synthesized from when the polymerase binds to DNA until it dissociates. Compared with Pfu DNA polymerase, which shows 20 bases of processivity, the higher processivity of the polymerase Excellent amplification ability without affecting PCR or affecting the GC rate or AT rate of the sequence. In the present invention, the processability measurement method is described in Appl. Environ. Microbiol. November 1997 Vol. 63, no. 11, p4504-4510.

More preferably, it is a weak DNA polymerase having strand displacement activity, preferably KlenTaq and Bst DNA polymerases for DNA polymerase belonging to Family A, and KOD, Phusion and Vent for DNA polymerases belonging to Family B. In the present invention, the strand displacement activity refers to a function of synthesizing a new DNA strand while dissociating hydrogen bonds of a double-stranded DNA serving as a template.

The presence or absence of strand displacement activity can be determined by adding a labeled probe having a 5'-end match to a reaction system containing FEN and performing qPCR. Since FEN nuclease cleaves only the flap structure, the strand displacement reaction is promoted, and the probe is cleaved only when the flap structure is taken. In qPCR, if an increase in fluorescence due to degradation of the labeled probe is confirmed, it is evaluated that there is a strand displacement ability. Although the strand displacement activity of KlenTaq and KOD is very weak, in the present invention, an increase in fluorescence is confirmed in the following measurement system, and it is regarded as having strand displacement ability. In the present invention, the flap structure indicates a triple-stranded structure of DNA, and indicates a structure of DNA in which a single strand or double strand protrudes to the 5 ′ side or 3 ′ side of a nick or gap portion on double stranded DNA. .

More specifically, KOD-Plus-Ver. 2 (Toyobo) attached 10 × PCR Buffer or rTaq DNA Polymerase (Toyobo) 10 × PCR Buffer, 1 × PCR Buffer, and 1.5 mM MgSO ₄ or MgCl ₂ , 0.2 mM dNTPs, β- 4 pmol of the primers described in SEQ ID NOS: 1 and 2 for amplifying actin, FAM 3 ′ which completely hybridizes with β-actin gene, 2 pmol probe modified with BHQ1 in 3 ′ (SEQ ID NO: 3), RNA equivalent to 5 ng Into 20 μl of the reaction solution containing the cDNA of 500 ng KOD FEN, add the enzyme to be determined for the presence or absence of strand displacement to 0.4 to 2.0 U. After the pre-reaction at 94 ° C. for 30 seconds, qPCR for recognizing FAM is performed with Light Cycler (registered trademark) 96 (Roche) on a schedule of 40 cycles of 94 ° C., 10 seconds → 60 ° C., 30 seconds. If an increase in fluorescence due to degradation of the labeled probe is confirmed, it is evaluated that there is a strand displacement ability.

The FEN nuclease in the present invention refers to flap structure-specific endoclease 1 and refers to an enzyme that recognizes the flap structure and cleaves nucleotides. Preferably, it is a thermostable FEN nuclease, and more preferable examples include thermococcus and pyrococcus FEN nuclease, but are not particularly limited.

The thermostable FEN nuclease in the present invention is a thermostable FEN nuclease that is stable even at a temperature of 60 ° C. or more. Specifically, the activity is maintained at 50% or more even after heating at 60 ° C. for 10 minutes. Shows the thermostable FEN nuclease to be produced. More preferably, the heat-resistant FEN nuclease is not particularly limited as long as the activity is maintained at 80% or more even when heated at 60 ° C. for 10 minutes. The method for measuring the activity of FEN nuclease is based on the method described in Japanese Patent No. 3018163.

The FEN and DNA polymerase in the present invention are characterized by acting separately without being fused. Thereby, the conventional problem that the reaction efficiency of DNA polymerase and the cleavage efficiency by FEN are reduced by using a fusion protein can be solved.

The labeled probe in the present invention is an oligonucleotide described in Patent Document 1 and the like, and preferably includes an oligonucleotide modified with a fluorescent dye. Methods used for detection such as modification of biotin and use of a radioisotope are used. There is no particular limitation. As the fluorescent substance, an oligonucleotide obtained by modifying a fluorescent substance such as FAM at the 5 'end and a quencher substance such as TAMRA at the 5' end is particularly preferable. There are various fluorescent materials modified in this case, such as FAM, VIC, HEX, and ROX, but there is no particular limitation. There are various quencher materials such as TAMRA and BHQ, but there is no particular limitation. The labeled probe in the present invention may have a mismatch inserted at the 5 'end or 3' end.

In the present invention, the 5 ′ end may be completely complementary, and since a flap structure formed by extension of DNA polymerase is cleaved and detected, a commercially available TaqMan® probe can also be detected. .

The detection of the target nucleic acid in the present invention may include a PCR inhibitor, and examples of the inhibitor include biological samples, but are not particularly limited as long as they are substances that inhibit PCR. The biological sample is not particularly limited as long as it is a sample collected from a living body. For example, it refers to animal and plant tissues such as body hair, nails, oral mucosa, body fluids such as blood, excrement such as feces and urine, cells, bacteria, viruses and the like. Body fluid includes blood and saliva, and cells include, but are not limited to, leukocytes separated from blood.

The detection method in the present invention may be used without purifying the nucleic acid in the biological sample. Here, purification is a method for separating contaminants such as tissues and cell walls of biological samples from DNA in biological samples, such as a method for separating DNA using phenol or phenol / chloroform, ion exchange, and the like. Examples thereof include a method of separating DNA using a resin, a glass filter, or a reagent having a protein aggregation action.

The detection method in the present invention may be detected by adding a biological sample to a nucleic acid amplification reaction solution without going through these purification steps. In the present invention, the “unpurified biological sample” also refers to the biological sample itself. However, a liquid biological sample diluted with a solvent such as water and a solid biological sample added to a solvent such as water and heat crushed are included. On the other hand, when nucleic acids to be amplified such as organs and cells are present in the tissue of the sample, an act of destroying the tissue to extract the nucleic acid (for example, destruction by physical treatment, using a surfactant, etc. Are not applicable to the purification in the present invention. Further, the act of diluting the sample obtained by the above method or the biological sample with a buffer solution does not correspond to the purification in the present invention.

In the present invention, if necessary, an antibody having the activity of suppressing the polymerase activity and / or 3'-5 'exonuclease activity of the thermostable DNA polymerase may be used. Examples of the antibody include a monoclonal antibody and a polyclonal antibody. This reaction composition is particularly effective for increasing the sensitivity of PCR and reducing nonspecific amplification.

Reagent for carrying out the nucleic acid amplification method The reagent for carrying out the detection of the present invention comprises:
(A) a DNA polymerase having no 5′-3 ′ exonuclease activity, and
(B) One that includes FEN nuclease in the reaction solution, uses a labeled probe, and cleaves the probe to determine the presence or absence of amplification. Other configurations are not particularly limited. The reagent includes a kit form.

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

Example 1
Preparation of FEN nuclease plasmid A plasmid containing the FEN nuclease gene derived from Thermococcus kodakaraensis KOD1 strain was prepared for use in the examples described below. The FEN nuclease gene was prepared by artificial synthesis with a His tag sequence added to the N-terminus (SEQ ID NO: 4) and cloned into pET23b (pFEN). The obtained plasmid was transformed into Escherichia coli BL21 (DE3) and used for enzyme preparation.

(Example 2)
Production of FEN nuclease The cells obtained in Example 1 were cultured as follows. First, 80 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 μg / mL ampicillin that was sterilized was dispensed into a 500 mL Sakaguchi flask. This medium was inoculated with Escherichia coli BL21 (DE3) (plasmid transformant) (using a test tube) previously cultured at 37 ° C. for 16 hours in 3 mL LB medium containing 100 μg / mL ampicillin at 30 ° C. Aeration culture was performed until OD660 was close to 0.5. Thereafter, sterilized IPTG was added to a final concentration of 0.5 mM, followed by aeration culture at 30 ° C. for 4 hours. The cells are collected from the culture solution by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, it is purified by nickel agarose chromatography. Finally, it is replaced with a storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 50% glycerin) to obtain FEN nuclease. It was.

(Example 3)
For use in the Examples Preparation <br/> below the FEN nuclease -KOD DNA polymerase fusion protein plasmid, plasmids containing genes of the fusion protein of FEN nuclease gene and KOD DNA polymerase derived from Thermococcus-kodakaraensis KOD1 strain Was made. Specifically, the linker described in Patent Document 4 and the sequence of KOD DNA polymerase were inserted into the C terminus of the FEN nuclease gene used in Example 1 using In Fusion (manufactured by TaKaRa), and the His tag sequence was inserted at the N-terminus. The given FEN-KOD (SEQ ID NO: 5) was cloned (pFEN-KOD). The obtained plasmid was transformed into Escherichia coli BL21 (DE3) and used for enzyme preparation.

Example 4
Production of FEN nuclease-KOD polymerase fusion protein The cells obtained in Example 3 were cultured as follows. First, 80 mL of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 μg / mL ampicillin that was sterilized was dispensed into a 500 mL Sakaguchi flask. This medium was inoculated with Escherichia coli BL21 (DE3) (plasmid transformant) (using a test tube) previously cultured at 37 ° C. for 16 hours in 3 mL LB medium containing 100 μg / mL ampicillin at 30 ° C. Aeration culture was performed until OD660 was close to 0.5. Thereafter, sterilized IPTG was added to a final concentration of 0.5 mM, followed by aeration culture at 30 ° C. for 4 hours. The cells are collected from the culture solution by centrifugation, suspended in 50 mL of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, it was purified by nickel agarose chromatography, and finally replaced with a storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 50% glycerin), and FEN nuclease-KOD. A polymerase fusion protein was obtained.

(Example 5)
Detection by labeled probe Comparison of detection was performed using a probe having a mismatch at the 5 ′ end (SEQ ID NO: 6) and a probe having no mismatch (SEQ ID NO: 3).
KOD-Plus-Ver. 2 (manufactured by Toyobo) attached with 10 × PCR Buffer, 1 × PCR Buffer, and 4 pmol of primers according to SEQ ID NOS: 1 and 2 for amplifying 1.5 mM MgSO 4, 0.2 mM dNTPs, β-actin, Examples A probe labeled with FAM and BHQ (SEQ ID NO: 6), in which 2 pmol of a sequence that does not hybridize to the target was added to 20 μl of a reaction solution containing 500 ng KOD FEN and 0.4 U KOD-Plus- prepared in 2; Alternatively, a probe (SEQ ID NO: 3) labeled with FAM and BHQ that completely hybridizes with the target is added, and total RNA of 25 ng, 2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, and 25 fg are used as templates. 94 ° C., 10 seconds after pre-reaction for 30 seconds QPCR which recognizes FAM was performed with Light Cycler (registered trademark) 96 (manufactured by Roche) on a schedule of 40 cycles of seconds → 60 ° C. and 30 seconds.

The qq Cq value and the results of the amplification curve are shown in FIG. The upper row uses a probe with no mismatch, and the lower row uses a probe with a mismatch. Although FEN nuclease is an enzyme that cleaves only the FLAP structure, a Cq value equal to or higher than that of a probe having a mismatch was obtained even with a probe having no mismatch at the 5 'end. This indicates that, due to the KOD strand displacement reaction, the extension progressed by floating the probe even if there was no mismatch sequence, and the FLAP structure was produced. It is thought that FEN recognized this FLAP structure and the probe was cleaved. Probes with no mismatch resulted in Cq variation due to detection at 250 fg and 25 fg, resulting in low detection efficiency. The present invention was able to detect a probe with no mismatch, and obtained a high result in terms of reaction efficiency. The same reaction was carried out with Phusion (manufactured by NEB) and Hemo KlenTaq (manufactured by NEB), which are known to have no 5′-3 ′ exonuclease, and the same probe as that with a mismatch was found even when there was no mismatch at the 5 ′ end. It was shown that the degree of detection was possible.

(Example 6)
Evaluation of FEN-KOD fusion protein It was evaluated whether FEN-KOD fusion protein can detect gene amplification with a probe (SEQ ID NO: 3) having no mismatch at the 5 'end.
KOD-Plus-Ver. 2 (manufactured by Toyobo) using 10 × PCR Buffer attached, 1 × PCR Buffer, and 4 pmol of the primers described in SEQ ID NOS: 1 and 2 for amplifying 1.5 mM MgSO 4, 0.2 mM dNTPs, β-actin, RNA corresponding to 25 ng In a 20 μl reaction solution containing the 50 ng FEN-KOD fusion protein prepared in Example 4 and a probe labeled with FAM, BHQ of SEQ ID NO: 3 that completely hybridizes with the target, or 1/30000 SYBR (registered trademark) Green is added, and after a pre-reaction of 94 ° C. for 30 seconds, 94 ° C., 10 seconds → 60 ° C., 30 seconds for 40 cycles, Light Cycler® 96 (manufactured by Roche) QPCR was performed.

The result of the qPCR amplification curve is shown in FIG. In the detection system of SYBR (registered trademark) Green shown in the upper part, an increase in fluorescence was confirmed, but in the probe system shown in the lower part, an increase in fluorescence could not be confirmed. It is considered that FEN and KOD movement deteriorated by using a fusion protein, and the probe could not be detected. Although it takes a lot of time for expression and purification, the method of the present invention in which FEN and DNA polymerase are separately added is easier than the method using a fusion protein in that an amount suitable for each reaction can be added. I think it can be built.

(Example 7)
Detection sensitivity comparison Using the FEN nuclease obtained in Example 2, the Taq reaction system was compared with the detection sensitivity.
The detection system of FEN is KOD-Plus-Ver. 2 (manufactured by Toyobo) attached with 10 × PCR Buffer, 1 × PCR Buffer, and 4 pmol of primers according to SEQ ID NOS: 1 and 2 for amplifying 1.5 mM MgSO 4, 0.2 mM dNTPs, β-actin, Examples 2 pmol of FAM and BHQ-labeled probe (SEQ ID NO: 3) were added to 20 μl of a reaction solution containing 500 ng KOD FEN nuclease and 0.4 U KOD-Plus- prepared in Step 2, and total RNA 25 ng, 2.5 ng, Using a cDNA equivalent to 250 pg, 25 pg, 2.5 pg, 250 fg, and 25 fg as a template, after a pre-reaction at 94 ° C. for 30 seconds, a schedule in which Light Cycler ( Registered trademark) 96 (manufactured by Roche) Recognition qPCR was performed.

In the Taq detection system, THUNDERBIRD qPCR Mix is used, and 4 pmol of the primers described in SEQ ID NOs: 1 and 2 for amplifying β-actin on 1 × MasterMix and a probe labeled with 2 pmol of FAM and BHQ (SEQ ID NO: 3) are included. Total RNA 25 ng, 2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 25 fg equivalent cDNA in 20 μl of reaction solution as a template, 94 ° C., 30 seconds pre-reaction, 94 ° C., 10 seconds → 60 ° C., QPCR which recognizes FAM was performed with Light Cycler 96 (Roche) on a schedule of repeating 30 seconds for 40 cycles.

The Cq value of qPCR and the result of the amplification curve are shown in FIG.
In the combination of FEN and KOD shown in the upper row, amplification was confirmed even from cDNA corresponding to 25 fg RNA, and better detection sensitivity than the Taq detection system shown in the lower row, which could only detect up to 250 fg, was confirmed. KOD is known to have detection sensitivity and PCR efficiency superior to Taq. The present invention makes it possible to detect a probe by utilizing the feature of KOD.

(Example 8)
Amplification of GC-rich target Using the FEN nuclease obtained in Example 2, the Taq reaction system was compared with the amplification of the GC-rich target.
The detection system of FEN is KOD-Plus-Ver. 2 (manufactured by Toyobo) using 10 × PCR Buffer attached to 1 × PCR Buffer, and 4 pmol of SEQ ID NOs: 7 and 8 that amplify β-globin exceeding 1.5% MgSO 4, 0.2 mM dNTPs and GC rate of 70%. A probe (SEQ ID NO: 9) labeled with 2 pmol of FAM and BHQ was added to 20 μl of a reaction solution containing the described primer, 500 ng KOD FEN nuclease prepared in Example 2, and 0.4 U KOD-Plus—, and gDNA was added. The template was subjected to qPCR for recognizing FAM with Light Cycler 96 (Roche) on a schedule of 94 cycles at 94 ° C. for 30 seconds followed by 40 cycles of 94 ° C., 10 seconds → 60 ° C., 30 seconds.

The Taq detection system uses THUNDERBIRD qPCR Mix and includes 4 pmol of the primers described in SEQ ID NOS: 7 and 8 for amplifying β-globin to 1 × MasterMix, and a probe labeled with 2 pmol of FAM and BHQ (SEQ ID NO: 9). Recognize FAM with Light Cycler 96 (Roche) on a schedule that repeats 40 cycles of 94 ° C, 10 seconds → 60 ° C, 30 seconds after pre-reaction of 94 ° C for 30 seconds using gDNA as a template in 20 µl of reaction solution QPCR was performed. 6 levels of gDNA were used as templates, 8.25 ng, 2 ng, 0.52 ng, 0.13 ng, 32 pg, and 8.1 pg, respectively.

The Cq value of qPCR and the result of the amplification curve are shown in FIG.
In the Taq detection system shown in the lower part, the target with a high GC rate cannot be amplified well, and the rise is delayed, whereas in the combination of FEN and KOD shown in the upper part, the rise is firmly confirmed up to 8.1 pg at the sixth level. It was done. It is confirmed that KOD and KlenTaq having high processivity are more resistant to amplification of sequences having such GC bias than normal Taq. By combining with FEN, a probe assay can be performed by utilizing the high performance of polymerase. Similarly, in the amplification of the untranslated region having a GC rate of 30% or less, KOD and KlenTaq can be amplified with high efficiency by combining with FEN, compared to normal Taq.

Example 9
Blood resistance Using the FEN nuclease obtained in Example 2, the Taq reaction system and amplification from blood were compared.
The detection system of FEN is KOD-Plus-Ver. 2 (manufactured by Toyobo) attached to 10 × PCR Buffer, 1 × PCR Buffer, and 4 pmol of primers according to SEQ ID NOs: 10 and 11 for amplifying 1.5 mM MgSO 4, 0.2 mM dNTPs, β-globin, Examples 2 pmol of FAM, BHQ-labeled probe (SEQ ID NO: 12) was added to 20 μl of a reaction solution containing 500 ng KOD FEN nuclease and 0.4 U KOD-Plus- prepared in step 2, and blood was used as a template at 94 ° C. After 30 seconds of pre-reaction, qPCR which recognizes FAM was performed with Light Cycler 96 (Roche) on a schedule of 40 cycles of 94 ° C., 10 seconds → 60 ° C., 30 seconds.
The Taq detection system uses THUNDERBIRD qPCR Mix and includes 4 pmol of the primers described in SEQ ID NOS: 10 and 11 for amplifying β-globin to 1 × MasterMix, and a probe labeled with 2 pmol of FAM and BHQ (SEQ ID NO: 12). Light Cycler (registered trademark) 96 (manufactured by Roche) is scheduled to repeat 40 cycles of 94 ° C, 10 seconds → 60 ° C, 30 seconds after pre-reaction at 94 ° C for 30 seconds with blood in 20 µl of reaction solution QPCR which recognizes FAM was performed. Each blood was added so that the reaction system contained 5%, 0.2%, and 0.05%.

The result of the qPCR amplification curve is shown in FIG.
Blood contains many substances that inhibit PCR, such as hemoglobin. In the Taq system shown in the lower part, amplification was possible only from a very thin blood such as 0.05%, but in the case of the combination of FEN and KOD shown in the upper part, amplification was possible even from a very dark blood of 5%.
The same reaction was also carried out with Phusion (manufactured by NEB) and Hemo KlenTaq (manufactured by NEB), which are known to be free of 5′-3 ′ exonuclease, with Phusion up to 5%. Hemo KlenTaq has been shown to detect up to 0.2%. Further, even the primer described in Example 8 having a high GC ratio could be detected from the same blood concentration.
Since it can be added to the qPCR reaction system without diluting the blood, it leads to a reduction in contamination and time. It can be used for diagnostic purposes.

The present invention is useful in biotechnology-related industries related to DNA synthesis, and particularly useful in diagnostic applications.

Claims (9)

A method for detecting a target nucleic acid having a GC rate or AT rate of 30% or less or 70% or more,
(A) a DNA polymerase having no 5′-3 ′ exonuclease activity, and
(B) Flap endonuclease (FEN)
A method for detecting the presence of a target nucleic acid by cleaving a labeled probe that hybridizes to a target nucleic acid to release a labeled fragment, and detecting or measuring the released labeled fragment. The method according to claim 1, wherein the labeled probe is an oligonucleotide modified with a fluorescent substance at the 5 'end and a quencher substance at the 3' end. The DNA polymerase having no 5′-3 ′ exonuclease activity is
(A) a DNA polymerase derived from family A that lacks 5′-3 ′ exonuclease activity, or
(B) selected from any of the DNA polymerases belonging to Family B, and
The method according to claim 1 or 2, wherein the process is a DNA polymerase having 50 or more bases. The DNA polymerase having no 5′-3 ′ exonuclease activity is
(A) a DNA polymerase derived from family A that lacks 5′-3 ′ exonuclease activity, or
(B) selected from any of the DNA polymerases belonging to Family B, and
The method according to any one of claims 1 to 3, which is a DNA polymerase having strand displacement ability. The method according to any one of claims 1 to 4, wherein the FEN nuclease is an FEN nuclease whose activity is maintained at 50% or more even when heated at 60 ° C or more for 10 minutes. The method according to claim 1, further comprising an unpurified biological sample in the reaction solution. The method according to claim 6, wherein the biological sample is any one selected from the group consisting of blood, stool, saliva, and urine. The reagent for performing the method for detecting the presence of the target nucleic acid according to any one of claims 1 to 7, wherein the following (a) and (b) are contained in the reaction solution.
(A) DNA polymerase having no 5′-3 ′ exonuclease activity (b) Flap endonuclease (FEN) A kit for performing a method for detecting the presence of a target nucleic acid comprising the reagent according to claim 8.