CN104928355A

CN104928355A - Method and kit thereof for detecting BRAF gene mutation

Info

Publication number: CN104928355A
Application number: CN201410104034.5A
Authority: CN
Inventors: 李跃; 高小祐; 孙景凤
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-03-19
Filing date: 2014-03-19
Publication date: 2015-09-23

Abstract

The invention discloses a method and kit thereof for detecting BARF gene mutation. The method comprises the following steps: (1) carrying out reactions in tube A, B, and C at the same time, carrying out BARF gene V600E and V600K mutation realtime quantitative PCR detection on a plasmid standard with a known mutation amount so as to obtain standard curves; (2) extracting and purifying the DNA of a sample, measuring the concentration, carrying out realtime quantitative PCR detection on the sample according to the tube A, B, and C reaction systems in the step (1), judging whether the gene mutation exists or not and the mutation amount according to the standard curves and the amplification fluorescence signals. The kit comprises a primer pair for detecting the No.15 exon mutation V600 E and V600K of BRAF gene, a probe, and a PNA repressing sequence. The provided method and kit can more precisely detect the No.15 exon mutation V600 E and V600K of BRAF gene, and the detection sensitivity is greatly improved.

Description

Method and kit for detecting BRAF gene mutation

Technical Field

The invention relates to a method for detecting gene mutation and a kit thereof, in particular to a method for detecting BRAF gene mutation and a kit thereof.

Background

The incidence of colorectal cancer increases year by year, and patients with advanced and postoperative recurrence need biological targeted therapy. It is clearly indicated in FDA and our country's postoperative treatment regimens that drug sensitive gene mutation detection is required when targeted drugs (cetuximab and panitumumab) targeting EGFR receptor antibodies are to be added in the implementation of first and second line chemotherapy regimens. The international large sample multi-center research shows that KRAS, BRAF, NRAS and PIK3CA gene mutation detection are carried out simultaneously, and serial diagnosis is carried out according to the sequence, so that the sensitivity of a patient to antibody-targeted drugs can be accurately predicted to the greatest extent (see the documents Roock WD, Claes B, Bernasconi D et al. effects of KRAS, BRAF, NRAS and PIK3CA multiple diagnosis of the efficacy of cetuximab plus chemotherapy in chemotherapy-reactive therapy cancer: aretroectotrophic consortium analysis. Lancet Oncol,2010,11: 753-62.).

The existing detection methods for BRAF gene mutation mainly comprise: direct Sequencing (Direct Sequencing), high resolution melting analysis (HRM), and amplification-dependent mutation system (ARMS). Direct Sequencing (Direct Sequencing) is based on PCR amplification technology, has a sensitivity lower than 10%, and has prevented its wide clinical application (refer to Lynch T, Bell D, Sordella R, et al. activating mutations in the epidermal growth factor amplification. N Engl J Med,2004,350(21):2129-2139.Paez J, Janne P, Lee J, et al. EGFR mutations in the stimulating center: Correlation with clinical expression thermal mutation. science,2004,304(4): 1497.). The high resolution melting analysis (HRM) is a principle of using different melting curves of DNA sequences with different lengths or different base compositions to complete mutation analysis of samples according to different melting points of amplified products after PCR amplification, and has a sensitivity of 1%, but requires a high-sensitivity precise Q-PCR instrument, and is mostly used in research, limiting clinical applications thereof (references Li LH, Tze KE, Chih CC, et al. characteristics and precision of KRAS, BRAF, and PIK3CA mutations in clinical analyzer by high-resolution analysis of Taiwave position. clinical Chimica Acta,413(2012) 1605-1611). Amplification disruption system (ARMS) is designed to make the 3' end sequence of primer respectively complement to wild and mutation, and make specific amplification and identification for mutant allele. In addition, Scorpion is a specific probe with scorpion structure, which comprises a PCR primer covalently connected with the 3' end, the primer only recognizes the mutation sequence and does not recognize the normal sequence, when the probe is connected with an amplicon (namely, the mutation sequence) for amplification in the instant PCR reaction, the fluorescent group is separated from the quenching group, and the fluorescence in the reaction reagent is enhanced. Scorpion in conjunction with ARMS technology (SARMS) can detect single mutations, with a high sensitivity of 0.1% for detection of known genes. The disadvantages of this method are: 1) each specific primer or probe can only identify one gene mutation type; 2) false positives due to single base mismatches may occur. The method is mainly used for various tissues including surgical, puncture or microscopic tissues, the detection can be completed within 2 hours, the commercialization degree of the kit is high, the clinical approval degree is high, the SFDA production lot is obtained from the national existing kit [ reference: Newton CR, Graham A, Heatintotal LE, et al.analysis of position tissue DNA ] the amplification recovery tissue system (ARMS). Nucleic Acids Res,1989,17(7):2503-2516.Marcin M. Machniki, Ela GM, Tomasz L, et al.ARMS-PCR for detection of BRAF V600E hotspot reaction in tissue with reaction-Time PCR-base, ACTA. POOCOCA, Vol. 60, Japanese No. 1-19. Forole tissue J.1998: 19. Fowlett-3: 2017. Forole tissue J.12. Fowlett-3-1267. Forole tissue of culture.

Peptide nucleic acid is a DNA analog whose backbone is substituted for the phosphodiester sugar of DNA by 2-aminoethylglycine, which has several advantages over traditional DNA probes: 1) PNA is very stable, the stability of correctly paired DNA/PNA is much higher than that of corresponding DNA/DNA, and PNA can not be used as substrate of Taq enzyme in PCR reaction process and can not be degraded by other enzymes. 2) Even a mismatch of only one base in the case of mismatched PNA/DNA will cause the melting temperature to drop by about 9-10 ℃. Peptide nucleic acids are currently used as a very useful molecular biological tool in the fields of disease diagnosis, treatment, etc. (see literature: Zhang Bing et al, systematic Comparison of PNA probes to DNA probes A systematic Complex PNA probes and DNA probes, macromolecular Notification 2006; 9: 62-68; EgholM, et al. PNA oligonucleotides to complementary oligonucleotides based on the Watson-Crick hydrogen-bonding mills, Nature,1993,365: 566-568.). Both Korean PANAGENE and U.S. PNA Bio Inc. use PNA as a clamp probe to repress amplification of wild DNA sequence, allow amplification detection of mutant sequence, and are used for screening of mutant sequence, and BRAF mutation detection kits were developed, but these detection methods cannot accurately detect the type of mutation and suffer from cross interference between different types of mutation.

The type and frequency of mutations posted by the Cosmic website for the BRAFV600 site are shown in table 1 below.

TABLE 1 BRAFV600 mutation types and frequencies released by Cosmic websites

The BRAF mutation detection in the current international clinical research mostly focuses on high-frequency V600E and V600K, which have clinical significance in tumor diagnosis and targeted drug guidance and are proved by a large number of clinical experiments, while the clinical significance of other mutation types (such as V600D, V600_ K601> E, V600M and V600R) is not reported accurately at present.

In conclusion, the V600E and V600K mutations occurring at high frequency in BRAF have clinical diagnosis significance, but the currently reported high-sensitivity PCR detection method lacks certain specificity, sensitivity and accuracy.

Disclosure of Invention

The invention aims to provide a method for detecting mutation (V600E and V600K) of the No.15 exon (exon 15) of a BRAF gene with high sensitivity and high specificity and a kit thereof.

In order to solve the above technical problems, the first aspect of the present invention provides a pair of primers for detecting mutations V600E and V600K in exon15 of BRAF gene, comprising:

1) an upstream sequence shown as SEQ ID NO.1 and a downstream sequence shown as SEQ ID NO. 2; or

2) A sequence at least 80% identical to the upstream sequence shown in SEQ ID NO.1 and a sequence at least 80% identical to the downstream sequence shown in SEQ ID NO. 2.

In a second aspect of the invention, there is provided a probe for detecting a wild-type BRAF gene, comprising: a Probe (Probe 1 wild) comprising the sequence shown in SEQ ID NO. 3.

Wherein, both ends of the Probe (Probe 1 wild) are respectively marked with a fluorescence reporter group (including FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA and the like) and a fluorescence quencher group (including MGB (minor groove binder) or TAMRA). For example, the probe may be a Taqman-MGB probe.

In a third aspect of the invention, there is provided a probe for detecting mutation V600E in exon15 of BRAF gene, comprising: a Probe comprising the sequence shown in SEQ ID NO.4 (Probe 2 mutant E1), a Probe comprising the sequence shown in SEQ ID NO.5 (Probe 3 mutant E2) or a Probe comprising the sequence shown in SEQ ID NO.6 (Probe 2 mutant E1E 2).

Wherein, both ends of the probe for detecting the BRAF gene exon15 mutation V600E can be respectively marked with a fluorescence reporter group (comprising FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA and the like) and a fluorescence quencher group (comprising MGB (minor groove binder) or TAMRA). For example, the probe may be a Taqman-MGB probe.

In a fourth aspect of the invention, there is provided a PNA (combined peptide nucleic acid) repressor sequence for detecting exon15 mutation V600E in BRAF gene, comprising: a PNA repression sequence (PNA-NBN-W) shown in SEQ ID NO.7 or a PNA repression sequence (PNA-HNN-K) shown in SEQ ID NO. 8;

SEQ ID NO.7：TTGGTCTAGCTACANBNAAATC; wherein,Nthe representation A, C, G or T is shown as,Bdenotes C, G or T, e.g.NBNSelected from the following combinations:

ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, ATA, ATT, ATC, ATG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, TTA, TTT, TTC, TTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, GGG, GTA, GTT, GTC, GTG, CCA, CCC, CCG, CGA, CGT, CGC, CGG, CTA, CTT, CTC, or CTG; the wild type V600 (GTG), the mutant V600M (ATG) and the mutant V600R (AGG) were mainly blocked.

Wherein AGG, ATG, GTG are wild or mutant sequences which have been reported to exist, and the mutant types are published according to the Cosmic website, and the AGG, ATG and GTG are unique closed sequences.

SEQ ID NO.8：TTGGTCTAGCTACAHNNAAATC; wherein,Hthe representation A, C or T is shown as,Ndenotes A, C, G or T, e.g.HNNSelected from the following combinations: AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, AGA, AGT, AGC, AGG, ACA, ACT, ACC, ACG, CAA, CAT, CAC, CAG, CTA, CTT, CTC, CTG, CGA, CGT, CGC, CGG, CCA, CCT, CCC, CCG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TTG, TGA, TGT, TGC, TGG, TCA, TCT, TCC or TCG; the mutations V600K (AAG), V600M (ATG) and V600R (AGG) were mainly blocked.

Wherein AAG, ATG and AGG are wild or mutant sequences which are reported to exist, and the mutant types are published according to the Coomic website, and are added to be unique closed sequences.

The PNA repression sequence for detecting the mutation V600E in exon15 of BRAF gene of the present invention is preferablyNBN（The method comprises the following steps: blocking wild type V600 (GTG), mutant V600M (ATG) and mutant V600R (AGG)) andHNN（mainly blocking the mutations V600K (AAG), V600M (ATG) and V600R (AGG)）While simultaneously applying. Namely, the PNA repression sequence for detecting the mutation V600E of the No.15 exon of BRAF gene comprises: PNA repression sequence (PNA-NBN-W) shown in SEQ ID NO.7 and PNA repression sequence (PNA-HNN-K) shown in SEQ ID NO.8, wherein the PNA repression sequence shown in SEQ ID NO.7NBNThe method comprises the following steps: GTG, ATG and AGG, of SEQ ID NO.8HNNThe method comprises the following steps: AAG, ATG and AGG.

Similarly, that is, the PNA repressor sequence for detecting exon15 mutation V600E in BRAF gene of the present invention also includes: the PNA repression sequence shown in SEQ ID NO.12 (for wild type) and the PNA repression sequence shown in SEQ ID NO.15 (for mutant K).

In a fifth aspect of the invention, there is provided a probe for detecting mutation V600K in exon15 of BRAF gene, comprising: a Probe comprising the sequence shown in SEQ ID NO.9 (Probe 3 mutant K).

Wherein, both ends of the probe for detecting the BRAF gene exon15 mutation V600K can be respectively marked with a fluorescence reporter group (comprising FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA and the like) and a fluorescence quencher group (comprising MGB (minor groove binder) or TAMRA). For example, the probe may be a Taqman-MGB probe.

In a sixth aspect of the invention, there is provided a degenerate probe for detecting mutations V600E and V600K in exon15 of the BRAF gene, comprising: a Probe (Probe 4 EK) comprising the sequence shown in SEQ ID NO.10 below;

SEQ ID NO. 10: TGGTCTAGCTACARARAA, respectively; wherein R is A or G.

Wherein, both ends of the facultative probe for detecting the mutation V600E and V600K of the No.15 exon of the BRAF gene can be respectively marked with a fluorescence reporter group (comprising FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA and the like) and a fluorescence quenching group (comprising MGB (minor groove binder) or TAMRA). For example, the degenerate probe may be a Taqman-MGB probe.

In a seventh aspect of the invention, there is provided a PNA repressor sequence for detecting exon15 mutation V600K in the BRAF gene, comprising: the PNA repression sequence shown in SEQ ID NO.11 (PNA-BNN-WE 1E 2);

SEQ ID NO.11：TTGGTCTAGCTACABNNAAATC; wherein,Bthe representation C, G or T is shown as,Ndenotes A, C, G or T, e.g.BNNSelected from the following combinations: CAA, CAT, CAC, CAG, CTA, CTT, CTC, CTG, CCA, CCC, CCG, CGA, CGT, CGC, CGG, GAA, GAT, GAC, GAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, GGG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TTG, TCA, TCT, TAC, TCG, TGA, TGT, TGC or TGG; the mutations V600E1 (GAG), V600E2(GAA) and wild type V600 (GTG) were mainly blocked.

Preference is given toBNNThe method comprises the following steps: GAG, GAA and GTG.

Similarly, that is, the PNA repressor sequence for detecting exon15 mutation V600K in BRAF gene of the present invention also includes: the PNA repression sequence shown in SEQ ID No.12 (for wild type), the PNA repression sequence shown in SEQ ID No.13 (for mutation E1) and the PNA repression sequence shown in SEQ ID No.14 (for mutation E2).

In an eighth aspect of the invention, there is provided a kit for detecting mutations V600E and V600K in exon15 of the BRAF gene, comprising:

the primer pair, the probe for detecting the wild type BRAF gene, the probe for detecting the mutation V600E of the No.15 exon of the BRAF gene or the merged probe for detecting the mutations V600E and V600K of the No.15 exon of the BRAF gene, the PNA repression sequence for detecting the mutation V600E of the No.15 exon of the BRAF gene, the probe for detecting the mutation V600K of the No.15 exon of the BRAF gene or the merged probe for detecting the mutations V600E and V600K of the No.15 exon of the BRAF gene and the PNA repression sequence for detecting the mutation V600K of the No.15 exon of the BRAF gene.

Preferably, the kit for detecting the mutation V600E of the No.15 exon of BRAF gene and V600K comprises:

1) a pair of primer pairs, the sequences of which are an upstream sequence shown as SEQ ID NO.1 and a downstream sequence shown as SEQ ID NO. 2;

2) a Probe (Probe 1 wild) comprising the sequence shown in SEQ ID NO. 3;

3) a Probe for detecting BRAF gene exon15 mutation V600E, which is a Probe containing a sequence shown in SEQ ID NO.4 (Probe 2 mutation E1), a Probe containing a sequence shown in SEQ ID NO.5 (Probe 3 mutation E2) or a Probe containing a sequence shown in SEQ ID NO.6 (Probe 2 mutation E1E 2); or

A degenerate Probe (Probe 4 EK) for detecting mutation V600E and V600K of the No.15 exon of BRAF gene;

4) the PNA repression sequence for detecting the mutation V600E of the No.15 exon of the BRAF gene is a PNA repression sequence (PNA-NBN-W) shown in SEQ ID NO.7 and a PNA repression sequence (PNA-HNN-K) shown in SEQ ID NO.8, or a combination of the PNA repression sequence (aiming at the wild type) shown in SEQ ID NO.12 and the PNA repression sequence (aiming at the mutation K) shown in SEQ ID NO. 15;

5) a Probe for detecting the mutation V600K of the No.15 exon of the BRAF gene, which is a Probe containing a sequence shown in SEQ ID NO.9 (Probe 3 mutation K) or a merged Probe (Probe 4 EK) for detecting the mutations V600E and V600K of the No.15 exon of the BRAF gene;

6) the PNA repression sequence for detecting the BRAF gene exon15 mutation V600K is the PNA repression sequence shown in SEQ ID NO.11 (PNA-BNN-WE 1E 2) or the combination of the PNA repression sequence shown in SEQ ID NO.12 (for wild type), the PNA repression sequence shown in SEQ ID NO.13 (for mutation E1) and the PNA repression sequence shown in SEQ ID NO.14 (for mutation E2);

in addition, the kit of the present invention may further include: BRAF gene wild type plasmid standard, BRAF gene V600E mutant type plasmid standard, BRAF gene V600K mutant type plasmid standard, PCR buffer solution, dNTPs, DNA polymerase and the like.

In a ninth aspect of the present invention, there is provided a method for real-time quantitative detection of mutations V600E and V600K in exon15 of BRAF gene (i.e. a method for qualitative and real-time quantitative detection of mutations V600E and V600K in exon15 of BRAF gene) using the above kit, comprising the steps of:

(1) carrying out the following A, B and C tube reactions at the same time, carrying out real-time quantitative PCR detection on V600E and V600K mutations of BRAF genes on plasmid standard products with known mutation quantities to obtain a standard curve;

wherein, the reaction system of the tube A comprises: GAG mutant plasmid standard of BRAF gene No.15 exon mutation V600E or GAA mutant plasmid standard of BRAF gene No.15 exon mutation V600E, the primer pair, a probe for detecting BRAF gene No.15 exon mutation V600E or a merged probe for detecting BRAF gene No.15 exon mutations V600E and V600K, a PNA repression sequence for detecting BRAF gene No.15 exon mutation V600E, enzyme buffer solution and water required by PCR amplification;

the reaction system of the tube B comprises: the kit comprises a plasmid standard product of BRAF gene No.15 exon mutation V600K, a primer pair, a probe for detecting BRAF gene No.15 exon mutation V600K or a merged probe for detecting BRAF gene No.15 exon mutations V600E and V600K, a PNA repression sequence for detecting BRAF gene No.15 exon mutation V600K, an enzyme buffer solution required for PCR amplification and water;

the reaction system of the tube C comprises: the kit comprises a BRAF gene wild type plasmid standard, the primer pair, a probe for detecting the wild type BRAF gene, an enzyme buffer solution required by PCR amplification and water;

(2) extracting and purifying sample DNA, measuring the concentration of the sample DNA, carrying out real-time quantitative PCR detection on the sample according to A, B and a C tube reaction system in the step (1), and distinguishing the existence and the mutation amount of the gene mutation according to a standard curve and an amplified fluorescent signal.

In the step (1), the gene sequence of GAG mutant plasmid standard product of BRAF gene No.15 exon mutation V600E is shown as SEQ ID NO.17, and the gene sequence of GAA mutant plasmid standard product of BRAF gene No.15 exon mutation V600E is shown as SEQ ID NO. 18;

the gene sequence of the plasmid standard product of BRAF gene No.15 exon mutation V600K is shown in SEQ ID NO. 19;

the gene sequence of the BRAF gene wild type plasmid standard is shown in SEQ ID NO. 16.

In the step (1), the reaction conditions of PCR are as follows: pre-denaturation at 95 ℃ for 10m, followed by denaturation at 95 ℃ for 15s, PNA binding at 70-75 ℃ for 15s, and annealing at 60 ℃ for 60s for 50 cycles.

In the step (2), the sample DNA comprises: DNA extracted from feces, tissues, interstitial fluid, cell lines and blood.

In the invention, in the detection of mutation of the BRAF gene exon15V600E, the mutation types detected by the probe are as follows:

GAG（E1）

GAA（E2）。

in the detection of mutation of BRAF gene exon15V600K, the mutation types detected by the probe are as follows: AAG (K).

TABLE 2 sequences

Therefore, the present invention can solve the following problems:

1. the PNA repression sequence is designed to inhibit the interference of the wild sequence corresponding to the mutation site on the specific amplification of the mutation sequence, namely, the non-specific amplification of the wild sequence is eliminated, and the specificity is improved.

2. Design PNA repression sequence, inhibit cross interference between different mutant sequences, namely eliminate cross reaction between different mutant sequences, improve specificity.

3. The merged PNA repressor sequence (e.g., SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.11, etc.) is designed such that the nucleotides to be detected at the same site are protected from the remaining 3 nucleotides.

4. The compatible Taqman-MGB probe (containing a sequence shown as SEQ ID NO.6 and a sequence shown as SEQ ID NO. 10) is designed, so that 3 mutant nucleotides which are different from a wild sequence at the same site can be detected.

5. And (3) comprehensively matching and assembling the PNA repression sequence and the Taqman-MGB probe. The degenerate PNA repressor sequence, the degenerate Taqman-MGB probe and the primer are assembled into a reaction system to realize the specific detection of a certain mutation site.

6. And (4) determining a reaction system. Including template, primer, PNA repression sequence, Taqman-MGB probe and enzyme buffer.

7. And (4) determining a PCR reaction program. Including denaturation, repression, determination of temperature and time of amplification and number of amplification cycles.

Based on the characteristic that peptide nucleic acid in the background technology can be complementarily combined with DNA, the invention combines the peptide nucleic acid with real-time quantitative PCR detection, designs fluorescence-labeled Taqman MGB probes respectively aiming at BRAF mutations with different properties, is applied to BRAF gene exon15 mutation detection, and can distinguish the mutation types of the BRAF genes exon15V600E and V600K.

The invention is realized by designing a Taqman MGB probe (comprising a degenerate probe containing a sequence shown as SEQ ID NO. 6) aiming at V600E, designing a Taqman MGB probe aiming at V600K, designing a degenerate Taqman MGB probe (comprising a sequence shown as SEQ ID NO. 10) aiming at simultaneously detecting V600E and V600K, designing a PNA repression sequence aiming at a wild type BRAF gene, designing PNA repression sequences (comprising a degenerate sequence) aiming at all other possible mutation types except the mutation type to be detected, and the like, namely designing a matched combined application form of the Taqman MGB probe aiming at a certain mutation type (comprising a merge type) and the corresponding repression PNA sequence, and the like.

The invention adopts a real-time quantitative PCR method combined with Peptide Nucleic Acid (PNA) to detect mutation and mutation quantity of the No.15 exon V600E and V600K of BRAF gene. Because PNA covers the wild sequence blocking the V600 site of BRAF gene and all other possible mutation types except the mutation type to be detected, and the variation of any site in the sequence causes the mismatch of PNA/DNA, so that the melting temperature (Tm) is obviously changed, the completely complementary sequence can be effectively repressed, the mismatched sequence is amplified, the mutation to be detected can be effectively amplified and detected, namely the wild type and the mutant can be distinguished, and the cross interference caused by other possible mutations can be inhibited. Therefore, the invention is a real-time quantitative detection method for mutation of the BRAF gene No.15 exon V600E and V600K, which has the advantages of simple operation, high sensitivity and high specificity.

The invention has the following beneficial effects:

1) improves the simple wild sequence blocking of the current PNA, designs the PNA repression sequence and increases the capacity of blocking other mutant sequences, and leads the detection of the mutation V600E and V600K of the No.15 exon of the BRAF gene to be more accurate.

2) The PNA blocking sequence is effectively combined with the Taqman MGB probe for application, and the detection sensitivity is greatly improved.

Drawings

The invention will be described in further detail with reference to the following detailed description and accompanying drawings:

FIG. 1 is the ABC tube test results of wild and mutant (3) plasmid reference samples, wherein FIG. 1-A is V600E mutant positive standard plasmid GAG, FIG. 1-B is V600E mutant positive standard plasmid GAA, FIG. 1-C is V600K mutant positive standard plasmid AAG, and FIG. 1-D is wild standard plasmid GTG;

FIG. 2 is the result of the detection of V600E mutation positive standard plasmid GAG serial gradient dilution sample, wherein, FIG. 2-A is ABC tube detection result; FIG. 2-B is a standard curve, wherein the slope of the standard curve is-3.951, Y-Inter (Y-intercept): 50.554, R²(coefficients of determination of the unary linear regression model): 0.975, eff% (amplification efficiency): 79.11;

FIG. 3 is a PCR amplification chart of detection of V600E mutation and V600K mutation of BRAF gene in 8 colorectal cancer tissue specimens, wherein FIG. 3-A is V600E mutation, and FIG. 3-B is V600K mutation.

Detailed Description

The invention is described in detail below with reference to the figures and examples. The following examples illustrate the present invention by performing real-time quantitative detection on plasmids containing mutation sequences at V600E and V600K sites of BRAF gene. It should be understood that this example is only for illustrating the present invention and is not intended to limit the scope of the present invention.

Example 1 detection of V600E and V600K mutant plasmid standards

V600E contains two nucleotide mutant sequences: GAG and GAA

The main reagent sources are as follows:

1.1 design of PNA

PNA was synthesized by Korea pangene and U.S. PNA Bio Inc.

PNA-NBN-W：NH₂-TTGGTCTAGCTACANBNAAATC-COOH; wherein N represents A, C, G or T, B represents C, G or T; the described merged sequences are mixed in a ratio of 1: 1.

PNA-HNN-K：NH₂-TTGGTCTAGCTACAHNNAAATC-COOH; wherein,Hthe representation A, C or T is shown as,Nrepresents A, C, G or T; the described merged sequences are mixed in a ratio of 1: 1.

PNA-BNN-E1E2：NH₂-TTGGTCTAGCTACABNNAAATC-COOH; wherein,Bthe representation C, G or T is shown as,Nrepresents A, C, G or T; the described merged sequences are mixed in a ratio of 1: 1.

1.2 design of Tagman MGB Probe

The Tagman MGB probe was synthesized by England Weiji (Shanghai) trade company, Inc.

Probe1 (Probe 1 wild): FAM (fluorescent marker) -TGGTCTAGCTACAGTGAA-MGB (minor groove binder)

Probe2 (Probe 2 mutant E1E 2): FAM-TGGTCTAGCTACAGARAA-MGB

Probe3 (Probe 3 mutant K): FAM-TGGTCTAGCTACAAAGAA-MGB

1.3PCR primers

The PCR primers were synthesized by England Weiji (Shanghai) trade Co., Ltd.

Upstream Primer sequence (Forward Primer):

5′-TCATGAAGACCTCACAGTAAAAATAGGT-3′（SEQ ID NO.1）

reverse Primer sequence (Reverse Primer):

5′-TGGGACCCACTCCATCGA-3′（SEQ ID NO.2）

1.4TaqMan Gene Expression Master Mix

purchased from applied biosystems, usa.

1.5BRAF Gene wild type and V600E and V600K mutant plasmid Standard construction

Wild type and mutant plasmid standards of the genus England are synthesized by England Weiji (Shanghai) trade company Limited and have the gene sequences of: and (2) wild:

CTGATAGGAAAATGAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCTTCATGAAG ACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGTGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCT（SEQ ID NO.16）

mutation 1-1gag (e):

CTGATAGGAAAATGAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGAGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCT（SEQ ID NO.17）

mutation 1-2gaa (e):

CTGATAGGAAAATGAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCTTCATGAAG ACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGAAAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCT（SEQ ID NO.18）

mutation 2aag (k):

CTGATAGGAAAATGAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAAAGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCT（SEQ ID NO.19）

diluting the 4 kinds of plasmid standard substances to 10 times⁶、10⁵、10⁴、10³、10²、10¹0 copies, each concentration gradient containing normal human genomic DNA125ng, from which a standard curve was prepared. Simultaneously, 10 of 4 plasmid standards are added⁵Copy number DNA was used as a reference sample.

1.6 real-time quantitative determination of reaction System and reaction conditions

The instrument is 7500 type real-time quantitative PCR instrument of ABI company. The sample detection is carried out simultaneously with three-tube reaction. PCR conditions were pre-denaturation at 95 ℃ for 10m, followed by denaturation at 95 ℃ for 15s, PNA binding at 70-75 ℃ for 15s, and annealing at 60 ℃ for 60s for 50 cycles.

Tube A: V600E detection, adding PNA, namely A-PNA +

And (B) tube: V600K detection, adding PNA, namely B-PNA +

C, pipe C: total V600 detection without PNA, namely C-PNA-

The A tube reaction system is shown in Table 3.

TABLE 3A tube reaction System

The reaction system in tube B is shown in Table 4.

TABLE 4B tube reaction System

The tube C reaction system is shown in Table 5.

TABLE 5C tube reaction System

Drawing a standard curve of quantitative detection:

the DNA standard template of the mutation 1-1 or the mutation 1-2 and the mutation 2 is diluted to 10 by continuous gradient⁶、10⁵、10⁴、10³、10²、10¹And 0 copy, and carrying out corresponding detection on the tube A or the tube B to prepare a standard curve.

1.7 detection of specificity of the detection method based on standards

Specific results for this detection method can be obtained from reference sample testing of plasmid standards.

As shown in figure 1 for the wild and mutant (3) plasmid reference samples of ABC tube test results, figure 1-a shows that V600E mutant positive standard plasmid GAG had a significant detection signal (smaller CT value) in tube a, no significant detection signal in tube B and a significant detection signal (smaller CT value) in tube C; indicated as the V600E mutation. FIG. 1-B shows that the results for the V600E mutation positive standard plasmid, GAA, are similar to those shown in FIG. 1-A; also indicated is the V600E mutation. Figure 1-C shows that V600K mutant positive standard plasmid AAG has no detection signal in tube a, significant detection signal in tube B (smaller CT value), and significant detection signal in tube C (smaller CT value); indicated as the V600K mutation. FIGS. 1-D show that wild standard plasmid GTG has no detection signal in tubes A and B and significant detection signal in tube C (smaller CT value); the V600 site was shown to be free of mutation and wild type.

In conclusion, the detection method can specifically detect the V600E mutation and the V600K mutation respectively, and is not interfered by the wild and other mutation types in the specific detection.

1.8 determination of sensitivity and quantitation of the detection method based on the Standard Curve

According to 10 for plasmid standards⁶、10⁵、10⁴、10³、10²、10¹And 0 copy of the sample is diluted by a continuous gradient, so that the detection sensitivity of the method can be obtained. The results of the serial gradient dilution of the V600E mutant positive standard plasmid GAG samples shown in fig. 3 indicate that the CT values of the samples detected by the method can be converted into quantitative values of copy number of the mutant genes in the samples by referring to the standard curve (as shown in fig. 2) (wherein, the relationship between the copy number and the CT value of the serial gradient dilution of the V600E mutant positive standard plasmid GAG samples is shown in table 6). The same is true. The same results were obtained with GAA and AAG.

TABLE 6 copy number versus CT value

Number of copies	CT value (A or B tube)
		2000	35.47
1000	36.75
		500	37.67
250	38.09
		125	40.09
63	41.01
		0	40.91

In addition, the reagents involved in this example include plasmids, probes, PNA sequences, primers, PCR reaction solutions, etc. which can be assembled into kits for detecting the mutations V600E and V600K.

Example 2 detection of the V600E and V600K mutations against colorectal cancer tissue specimens

2.1 specimen collection:

collecting 8 cases of cancer (cancer diagnosis is based on clinical pathological diagnosis including pathological diagnosis) of fresh colorectal cancer tissue specimen excised by operation, wherein the tissue is about 1 mg.

2.2DNA extraction method:

a commercially available Qiangen genome DNA extraction kit was selected, the genome DNA was extracted according to the instructions, and 100. mu.l of TE was used to dissolve the DNA, which was then stored at-80 ℃.

2.3DNA concentration determination

The concentration was measured using a NanoDrop ND-1000 full wavelength UV/Vis scanning spectrophotometer.

2.4 samples for V600E and V600K mutation detection

Sample DNA125ng was taken and subjected to ABC three-tube assay as described in example 1, method 1.6.

2.5 analysis of results

Referring to the 1.7 specific assay in example 1 and the results in FIG. 3, the A tube V600E mutation test was found to be1 positive and the B tube V600K was not found to be positively amplified.

The result judges that 1 BRAF V600E mutation exists in 8 colorectal cancer tissue specimens, and the rest are wild types.

Claims

1. A pair of primers for detecting mutation V600E and mutation V600K of the No.15 exon of BRAF gene is characterized by comprising:

2. A probe for detecting a wild-type BRAF gene, comprising: a probe comprising the sequence shown in SEQ ID NO. 3.

3. The probe of claim 2, wherein: the two ends of the probe are respectively marked with a fluorescence reporter group and a fluorescence quenching group;

wherein the fluorescent reporter group comprises: FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA;

the fluorescence quenching group comprises: MGB or TAMRA.

4. A probe for detecting mutation V600E of the No.15 exon of BRAF gene, which is characterized by comprising: a probe comprising the sequence shown in SEQ ID NO.4, a probe comprising the sequence shown in SEQ ID NO.5 or a probe comprising the sequence shown in SEQ ID NO. 6.

5. The probe of claim 4, wherein: the two ends of the probe are respectively marked with a fluorescence reporter group and a fluorescence quenching group;

the fluorescence quenching group comprises: MGB or TAMRA.

6. A PNA repressor sequence for detecting exon15 mutation V600E in the BRAF gene, comprising: a PNA repression sequence as shown in SEQ ID No.7 or a PNA repression sequence as shown in SEQ ID No. 8;

SEQ ID NO. 7: TTGGTCTAGCTACANNBNAAATC; wherein N represents A, C, G or T, B represents C, G or T;

SEQ ID NO. 8: TTGGTCTAGCTACAHNNAAATC, respectively; wherein H represents A, C or T, and N represents A, C, G or T.

7. PNA repression sequence according to claim 6, characterized in that: the NBN in SEQ ID NO.7 is selected from the following combinations:

ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, ATA, ATT, ATC, ATG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, TTA, TTT, TTC, TTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, GGG, GTA, GTT, GTC, GTG, CCA, CCC, CCG, CGA, CGT, CGC, CGG, CTA, CTT, CTC or CTG;

the HNN in SEQ ID NO.8 is selected from the following combinations:

AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, AGA, AGT, AGC, AGG, ACA, ACT, ACC, ACG, CAA, CAT, CAC, CAG, CTA, CTT, CTC, CTG, CGA, CGT, CGC, CGG, CCA, CCT, CCC, CCG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TTG, TGA, TGT, TGC, TGG, TCA, TCT, TCC or TCG.

8. PNA repression sequence according to claim 6, characterized in that: the PNA repression sequence for detecting the mutation V600E of the No.15 exon of BRAF gene comprises: a PNA repression sequence shown by SEQ ID NO.7 and a PNA repression sequence shown by SEQ ID NO. 8;

wherein the NBN in SEQ ID NO.7 comprises: GTG, ATG and AGG;

the HNN in SEQ ID NO.8 comprises: AAG, ATG and AGG.

9. PNA repression sequence according to claim 6, characterized in that: the PNA repression sequence for detecting the mutation V600E of the No.15 exon of BRAF gene comprises: the PNA repression sequence as shown in SEQ ID NO.12 and the PNA repression sequence as shown in SEQ ID NO. 15.

10. A probe for detecting mutation V600K of the No.15 exon of BRAF gene, which is characterized by comprising: a probe comprising the sequence shown in SEQ ID NO. 9.

11. A probe as claimed in claim 10, wherein: the two ends of the probe are respectively marked with a fluorescence reporter group and a fluorescence quenching group;

the fluorescence quenching group comprises: MGB or TAMRA.

12. A degenerate probe for detecting mutations V600E and V600K in exon15 of BRAF gene, comprising: a probe comprising a sequence shown in SEQ ID NO. 10;

SEQ ID NO. 10: TGGTCTAGCTACARARAA, respectively; wherein R is A or G.

13. A probe as claimed in claim 12, wherein: the two ends of the probe are respectively marked with a fluorescence reporter group and a fluorescence quenching group;

the fluorescence quenching group comprises: MGB or TAMRA.

14. A PNA repressor sequence for detecting exon15 mutation V600K in the BRAF gene, comprising: the PNA repression sequence shown as SEQ ID No. 11;

SEQ ID NO. 11: TTGGTCTAGCTACACNNACT; wherein B represents C, G or T, and N represents A, C, G or T.

15. The PNA repression sequence of claim 14, wherein: the BNN in SEQ ID NO.11 is selected from the following combinations:

CAA, CAT, CAC, CAG, CTA, CTT, CTC, CTG, CCA, CCC, CCG, CGA, CGT, CGC, CGG, GAA, GAT, GAC, GAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, GGG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TTG, TCA, TCT, TAC, TCG, TGA, TGT, TGC or TGG.

16. The PNA repression sequence of claim 14, wherein: the PNA repression sequence comprising: a PNA repression sequence as shown in SEQ ID NO.12, a PNA repression sequence as shown in SEQ ID NO.13 and a PNA repression sequence as shown in SEQ ID NO. 14.

17. A kit for detecting mutations V600E and V600K in exon15 of the BRAF gene, comprising:

the primer pair of claim 1, the probe for detecting wild-type BRAF gene of claim 2, the probe for detecting exon15 mutation V600E of BRAF gene of claim 4 or the degenerate probe for detecting exon15 mutations V600E and V600K of BRAF gene of claim 12, the PNA repressor sequence for detecting exon15 mutation V600E of BRAF gene of claim 6, the probe for detecting exon15 mutation V600K of BRAF gene of claim 10 or the degenerate probe for detecting exon15 mutation V600K of BRAF gene of claim 12 or the degenerate probe for detecting exon15 mutations V600E and V600K of BRAF gene of claim 14, and the PNA repressor sequence for detecting exon15 mutation V600K of BRAF gene of claim 14.

18. The kit of claim 17, wherein: the kit comprises:

2) a probe comprising a sequence shown in SEQ ID NO. 3;

3) the probe for detecting the mutation V600E of the 15 th exon of the BRAF gene is a probe containing a sequence shown in SEQ ID NO.4, a probe containing a sequence shown in SEQ ID NO.5 or a probe containing a sequence shown in SEQ ID NO. 6; or

A degenerate probe for detecting mutation of exon15 of BRAF gene V600E and V600K;

4) the PNA repression sequence for detecting the mutation V600E of the No.15 exon of the BRAF gene is a PNA repression sequence shown in SEQ ID NO.7 and a PNA repression sequence shown in SEQ ID NO.8, or a combination of the PNA repression sequence shown in SEQ ID NO.12 and the PNA repression sequence shown in SEQ ID NO. 15;

5) the probe for detecting the mutation V600K of the No.15 exon of the BRAF gene is a probe containing a sequence shown in SEQ ID NO.9 or a merged probe for detecting the mutations V600E and V600K of the No.15 exon of the BRAF gene;

6) the PNA repression sequence for detecting the mutation V600K of the No.15 exon of the BRAF gene is the PNA repression sequence shown in SEQ ID NO.11 or a combination of the PNA repression sequence shown in SEQ ID NO.12, the PNA repression sequence shown in SEQ ID NO.13 and the PNA repression sequence shown in SEQ ID NO. 14.

19. The kit of claim 17, wherein: the kit further comprises: BRAF gene wild type plasmid standard, BRAF gene V600E mutant type plasmid standard, BRAF gene V600K mutant type plasmid standard, PCR buffer solution, dNTPs and DNA polymerase.

20. A method for real-time quantitative detection of mutation V600E and V600K of the 15 th exon of BRAF gene by using the kit of claim 17, which is characterized by comprising the following steps:

21. The method of claim 20, wherein: in the step (1), the gene sequence of GAG mutant plasmid standard product of BRAF gene No.15 exon mutation V600E is shown in SEQ ID NO.17, and the gene sequence of GAA mutant plasmid standard product of BRAF gene No.15 exon mutation V600E is shown in SEQ ID NO. 18;

the gene sequence of the BRAF gene wild type plasmid standard is shown in SEQ ID NO. 16;

in the step (1), the reaction conditions of PCR are as follows: pre-denaturation at 95 ℃ for 10m, subsequent denaturation at 95 ℃ for 15s, PNA binding at 70-75 ℃ for 15s, and annealing at 60 ℃ for 60s for 50 cycles;