CN104928355A - Method and kit thereof for detecting BRAF gene mutation - Google Patents
Method and kit thereof for detecting BRAF gene mutation Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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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
Technical field
The present invention relates to a kind of method and the test kit thereof that detect transgenation, particularly relate to a kind of method and the test kit thereof that detect BRAF gene mutation.
Background technology
Colorectal cancer incidence rate increases year by year, late period and postoperative recurrence needs of patients Biological target therapy.Explicitly point out in U.S. FDA and China's post operative colo-rectal cancer treatment plan, carrying out in a line and second-line chemotherapy scheme implementation, when intending adding targeting EGFR receptor antibody targeted drug (Cetuximab and Victibix), need susceptibility detection in Gene Mutation be carried out.International large sample multicenter study shows, carry out KRAS simultaneously, BRAF, NRAS, PIK3CA detection in Gene Mutation, and diagnosis of connecting in this order, can to greatest extent Accurate Prediction patient for the susceptibility of antibody target medicine (see document Roock WD, Claes B, Bernasconi D et al.Effects of KRAS, BRAF, NRAS and PIK3CA mutationson the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer:aretrospective consortium analysis.Lancet Oncol, 2010, 11:753-62.).
Detection method at present for BRAF gene mutation mainly contains: direct sequencing (Direct Sequencing), high resolving power solubility curve analytical technology (high resolution melting analysis, and amplification refractory mutation system technology (amplification refractory mutationsystem, ARMS) HRM).Direct sequencing (Direct Sequencing) with pcr amplification technology for core, its sensitivity is lower is greater than 10%, hinder its wide clinical application (reference Lynch T, Bell D, Sordella R, et al.Activating mutations in the epidermal growth factorreceptor underlying responsiveness of non – small-cell lung cancer to gefitinib.N Engl J Med, 2004, 350 (21): 2129-2139.Paez J, Janne P, Lee J, et al.EGFR mutations in lung cancer:Correlation with clinicalresponse to gefitinib therapy.Science, 2004, 304 (4): 1497-1500.).High resolving power solubility curve analytical technology (high resolutionmelting analysis, HRM) be the principle utilizing the DNA sequence dna solubility curve of different lengths or different based composition different, according to the different complete paired samples mutation analysis of the fusing point of amplified production after pcr amplification, its sensitivity can reach 1%, but need highly sensitive accurate Q-PCR instrument, be used in research, limit its clinical application (reference Li LH, Tze KE, Chih CC, et al.Characteristics and prevalence ofKRAS, BRAF, and PIK3CA mutations in colorectal ancer by high-resolution melting analysis in Taiwanesepopulation.Clinica Chimica Acta, 413 (2012) 1605 – 1611).Amplification refractory mutation system technology (amplification refractorymutation system, ARMS) is the wild and sudden change complementation respectively of design primer 3 ' terminal sequence, and the allelotrope for sudden change carries out specific amplification and qualification.Scorpions is the specific probe of scorpion-like structure in addition, comprise one and 3 ' PCR primer of holding covalency to be connected, primer only identifies mutant nucleotide sequence, and nonrecognition normal sequence, in instant PCR reaction, when probe to be connected with amplicon (i.e. mutant nucleotide sequence) increase time, fluorophor is separated with quenching group, and in reaction reagent, fluorescence strengthens.Scorpions and ARMS technology connection and application (SARMS) can detect single sudden change, and for the detection of known, SARMS has the susceptibility 0.1% of height.The shortcoming of the method is: 1) every species-specific primer or probe can only be identified for a certain gene mutation type; 2) false positive because single base mispairing causes may be there is.The method is mainly used in various organization, comprise operation, puncture or microscopy organize class, within 2 hours, detection can be completed, test kit commercialization degree is high, the clinical degree of recognition is high, domestic existing test kit obtains SFDA and produces certification [reference Newton CR, Graham A, Heptinstall LE, et al.Analysis ofany point mutation in DNA:the amplification refractory mutation system (ARMS) .Nucleic Acids Res, 1989, 17 (7): 2503-2516.Marcin M.Machnicki, Eliza GM, Tomasz L, et al.ARMS-PCR for detection of BRAF V600E hotspotmutation in comparison with Real-Time PCR-based techniques.ACTA BIOCHIMICA POLONICA, Vol. 60, No1/2013:57 – 64.Fox JC, et al.The detection of K-ras mutations in colorectal cancer using theamplification-refractory mutation system, Br J Cancer1998, 77:1267-74.].
Peptide nucleic acid(PNA) is a kind of DNA analogue, its skeleton instead of the phosphodiester sugar of DNA by 2-aminoethyl glycine, it has many advantages than traditional DNA probe: 1) PNA ten points stablizes, the stability of the DNA/PNA of correct pairing is far away higher than corresponding DNA/DNA, simultaneously in PCR reaction process, PNA not can be used as the substrate of Taq enzyme, also can not degrade by other enzymes.2) for the PNA/DNA of mispairing, even only have the mispairing of a base, its melting temp decline about 9-10 DEG C can also be caused.At present, peptide nucleic acid(PNA) is applied (see document: Zhang Bingbo etc. in fields such as the diagnosis of disease, treatments as a kind of very useful biology tool, the systematic comparison A Systemic Comparison between PNA Probe and DNA Probe of PNA probe and DNA probe, " polymer circular " 2006; 9:62-68; Egholm M, et al.PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules, Nature, 1993,365:566 ~ 568.).PANAGENE company of Korea S and PNA Bio Inc. company of the U.S. all apply PNA as clamping down on probe, check the amplification of wild DNA sequence dna, mutant nucleotide sequence is made to be able to augmentation detection, for the examination of mutant nucleotide sequence, have developed BRAF mutation detection kit, but these detection methods accurately can not detect mutation type, and be subject to the cross interference between different mutation type.
For BRAFV600 site, Cosmic website orientation mutation type and frequency as shown in table 1 below.
The BRAFV600 mutation type of table 1Cosmic website orientation and frequency
Current international clinical study is carried out BRAF abrupt climatic change and is focused mostly in V600E and V600K of high frequency appearance, its clinical meaning that there is diagnosing tumor and instruct target medication, and clinical experiment confirms in a large number, and for other mutation types (as V600D, V600_K601>E, V600M and V600R) clinical meaning at present without accurate report.
In sum, V600E and the V600K sudden change that BRAF high frequency occurs has clinical diagnosis meaning, but the highly sensitive PCR detection method reported at present, lack certain specificity, susceptibility and accuracy.
Summary of the invention
The technical problem to be solved in the present invention is to provide method and the test kit thereof of the detection BRAF gene the 15th exon (exon15) sudden change (V600E and V600K) of a kind of highly sensitive and high specific degree.
For solving the problems of the technologies described above, a first aspect of the present invention, providing a pair for detecting the primer pair of BRAF gene the 15th exon sudden change V600E and V600K, comprising:
1) upstream sequence as shown in SEQ ID NO.1 and the downstream sequence as shown in SEQ ID NO.2; Or
2) at least identical with the upstream sequence 80% shown in SEQ ID NO.1 sequence and at least identical with the downstream sequence 80% shown in SEQ ID NO.2 sequence.
A second aspect of the present invention, providing a kind of probe for detecting wild-type BRAF gene, comprising: the probe (Probe1 is wild) comprising sequence shown in SEQ ID NO.3.
Wherein, the two ends of described probe (Probe1 is wild) are also marked with fluorescent reporter group (comprising: FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA etc.) and fluorescent quenching group (comprising: MGB(minor groove binder) or TAMRA) respectively.As this probe can be a kind of Taqman-MGB probe.
A third aspect of the present invention, a kind of probe for detecting BRAF gene the 15th exon sudden change V600E being provided, comprising: the probe (Probe2 suddenly change E1) comprising sequence shown in SEQID NO.4, the probe (Probe3 suddenly change E2) comprising sequence shown in SEQ ID NO.5 or the probe (Probe2 suddenly change E1E2) comprising sequence shown in SEQ ID NO.6.
Wherein, the described two ends of probe for detecting BRAF gene the 15th exon sudden change V600E also can be marked with fluorescent reporter group (comprising: FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA etc.) and fluorescent quenching group (comprising: MGB(minor groove binder) or TAMRA) respectively.As this probe can be a kind of Taqman-MGB probe.
A fourth aspect of the present invention, a kind of PNA(for detecting BRAF gene the 15th exon sudden change V600E is provided to combine peptide nucleic acid(PNA)) repressor sequences, comprising: the PNA repressor sequences (PNA-NBN-W) shown in SEQ ID NO.7 or the PNA repressor sequences (PNA-HNN-K) shown in SEQ ID NO.8;
SEQ ID NO.7:TTGGTCTAGCTACA
nBNaAATC; Wherein,
nrepresent A, C, G or T,
brepresent C, G or T, as
nBNbe selected from following combination:
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, CCT, CCC, CCG, CGA, CGT, CGC, CGG, CTA, CTT, CTC or CTG; Mainly enclose wild-type V600(GTG), sudden change V600M(ATG) and suddenly change V600R(AGG).
Wherein, AGG, ATG, GTG* have reported the wild of existence or mutant nucleotide sequence, and with reference to above-mentioned Cosmic website orientation mutation type, adding * is unique sequence closed.
SEQ ID NO.8:TTGGTCTAGCTACA
hNNaAATC; Wherein,
hrepresent A, C or T,
nrepresent A, C, G or T, as
hNNbe selected from following combination: 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; Mainly enclose sudden change V600K(AAG), sudden change V600M(ATG) and suddenly change V600R(AGG).
Wherein, AAG*, ATG, AGG have reported the wild of existence or mutant nucleotide sequence, and with reference to above-mentioned Cosmic website orientation mutation type, adding * is unique sequence closed.
PNA repressor sequences for detecting BRAF gene the 15th exon sudden change V600E of the present invention is preferred
nBN(comprise: enclose wild-type V600(GTG), sudden change V600M(ATG) and suddenly change V600R(AGG)) and
hNN(mainly enclose sudden change V600K(AAG), sudden change V600M(ATG) and suddenly change V600R(AGG)
)while application.Namely for detecting the PNA repressor sequences of BRAF gene the 15th exon sudden change V600E, comprise: the PNA repressor sequences (PNA-NBN-W) shown in SEQ ID NO.7 and the PNA repressor sequences (PNA-HNN-K) shown in SEQ IDNO.8, wherein, in SEQ ID NO.7
nBNcomprise: GTG, ATG and AGG, in SEQ ID NO.8
hNNcomprise: AAG, ATG and AGG.
In like manner, that is, PNA repressor sequences for detecting BRAF gene the 15th exon sudden change V600E of the present invention, also comprises: the PNA repressor sequences (for wild-type) as shown in SEQ ID NO.12 and the PNA repressor sequences as shown in SEQ ID NO.15 (for sudden change K).
A fifth aspect of the present invention, providing a kind of probe for detecting BRAF gene the 15th exon sudden change V600K, comprising: the probe (Probe3 suddenly change K) comprising sequence shown in SEQID NO.9.
Wherein, the described two ends of probe for detecting BRAF gene the 15th exon sudden change V600K also can be marked with fluorescent reporter group (comprising: FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA etc.) and fluorescent quenching group (comprising: MGB(minor groove binder) or TAMRA) respectively.As this probe can be a kind of Taqman-MGB probe.
A sixth aspect of the present invention, providing a kind of merger probe for detecting BRAF gene the 15th exon sudden change V600E and V600K, comprising: the probe (Probe4EK) comprising sequence shown in following SEQ ID NO.10;
SEQ ID NO.10:TGGTCTAGCTACARARAA; Wherein, R is A or G.
Wherein, the described two ends of merger probe for detecting BRAF gene the 15th exon sudden change V600E and V600K also can be marked with fluorescent reporter group (comprising: FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA etc.) and fluorescent quenching group (comprising: MGB(minor groove binder) or TAMRA) respectively.As this merger probe can be a kind of Taqman-MGB probe.
A seventh aspect of the present invention, providing a kind of PNA repressor sequences for detecting BRAF gene the 15th exon sudden change V600K, comprising: the PNA repressor sequences (PNA-BNN-WE1E2) shown in SEQ ID NO.11;
SEQ ID NO.11:TTGGTCTAGCTACA
bNNaAATC; Wherein,
brepresent C, G or T,
nrepresent A, C, G or T, as
bNNbe selected from following combination: CAA, CAT, CAC, CAG, CTA, CTT, CTC, CTG, CCA, CCT, 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, TCC, TCG, TGA, TGT, TGC or TGG; Mainly enclose sudden change V600E1(GAG), sudden change V600E2 (GAA) and wild-type V600(GTG).
Preferably
bNNcomprise: GAG, GAA and GTG.
In like manner, that is, PNA repressor sequences for detecting BRAF gene the 15th exon sudden change V600K of the present invention, also comprises: the PNA repressor sequences (for wild-type) as shown in SEQ ID NO.12, the PNA repressor sequences (for sudden change E1) as shown in SEQ ID NO.13 and the PNA repressor sequences (for the E2 that suddenlys change) as shown in SEQ ID NO.14.
A eighth aspect of the present invention, providing a kind of test kit for detecting BRAF gene the 15th exon sudden change V600E and V600K, comprising:
Above-mentioned primer pair, for detecting the probe of wild-type BRAF gene, for detecting the probe of BRAF gene the 15th exon sudden change V600E or the merger probe for detecting BRAF gene the 15th exon sudden change V600E and V600K, for detecting the PNA repressor sequences of BRAF gene the 15th exon sudden change V600E, for detecting the probe of BRAF gene the 15th exon sudden change V600K or the merger probe for detecting BRAF gene the 15th exon sudden change V600E and V600K, and for detecting the PNA repressor sequences of BRAF gene the 15th exon sudden change V600K.
Preferably, the described test kit for detecting BRAF gene the 15th exon sudden change V600E and V600K, comprising:
1) pair of primers pair, its sequence is the such as upstream sequence shown in SEQ ID NO.1 and the downstream sequence as shown in SEQ ID NO.2;
2) probe (Probe1 is wild) of sequence shown in SEQ ID NO.3 is comprised;
3) for detecting the probe of BRAF gene the 15th exon sudden change V600E, its for comprise sequence shown in SEQ ID NO.4 probe (Probe2 suddenly change E1), comprise the probe (Probe3 suddenly change E2) of sequence shown in SEQ ID NO.5 or comprise the probe (Probe2 suddenly change E1E2) of sequence shown in SEQ ID NO.6; Or
For detecting the merger probe (Probe4EK) of BRAF gene the 15th exon sudden change V600E and V600K;
4) for detecting the PNA repressor sequences of BRAF gene the 15th exon sudden change V600E, it is the PNA repressor sequences (PNA-HNN-K) shown in the PNA repressor sequences (PNA-NBN-W) shown in SEQ ID NO.7 and SEQ ID NO.8, or the combination be made up of the PNA repressor sequences (for sudden change K) shown in the PNA repressor sequences (for wild-type) shown in SEQ ID NO.12 and SEQ ID NO.15;
5) for detecting the probe of BRAF gene the 15th exon sudden change V600K, it is for comprising the probe (Probe3 suddenly change K) of sequence shown in SEQ ID NO.9 or suddenling change the merger probe (Probe4EK) of V600E and V600K for detecting BRAF gene the 15th exon;
6) for detecting the PNA repressor sequences of BRAF gene the 15th exon sudden change V600K, it is the PNA repressor sequences (PNA-BNN-WE1E2) shown in SEQ ID NO.11 or the combination that is made up of the PNA repressor sequences (for wild-type) such as shown in SEQ ID NO.12, the PNA repressor sequences as shown in SEQ IDNO.13 (for sudden change E1) and PNA repressor sequences as shown in SEQ ID NO.14 (for the E2 that suddenlys change);
In addition, test kit of the present invention, also can comprise: BRAF gene wild plasmid standard substance, BRAF gene V600E mutant plasmids standard substance, BRAF gene V600K mutant plasmids standard substance, PCR damping fluid, dNTPs and archaeal dna polymerase etc.
A ninth aspect of the present invention, a kind of method (i.e. the method for a kind of energy qualitative and Real_time quantitative detection BRAF gene the 15th exon V600E and V600K sudden change) utilizing mentioned reagent box to carry out Real_time quantitative detection BRAF gene the 15th exon sudden change V600E and V600K is provided, comprises step:
(1) carry out following A, B and C tube reaction simultaneously, the real-time quantitative PCR that the plasmid standard of known mutations amount carries out suddenling change about V600E and V600K of BRAF gene is detected, draws typical curve;
Wherein, the reaction system of A pipe comprises: the GAA mutant plasmids standard substance of BRAF gene the 15th exon sudden change GAG mutant plasmids standard substance of V600E or BRAF gene the 15th exon sudden change V600E, above-mentioned primer pair, for detect BRAF gene the 15th exon sudden change V600E probe or for detect BRAF gene the 15th exon sudden change V600E and V600K merger probe, for detecting enzyme buffer liquid needed for the PNA repressor sequences of BRAF gene the 15th exon sudden change V600E, pcr amplification and water;
The reaction system of B pipe comprises: BRAF gene the 15th exon sudden change plasmid standard of V600K, above-mentioned primer pair, for detect BRAF gene the 15th exon sudden change V600K probe or for detect BRAF gene the 15th exon sudden change V600E and V600K merger probe, for detecting enzyme buffer liquid needed for the PNA repressor sequences of BRAF gene the 15th exon sudden change V600K, pcr amplification and water;
The reaction system of C pipe comprises: BRAF gene wild plasmid standard substance, above-mentioned primer pair, for detecting enzyme buffer liquid needed for the probe of wild-type BRAF gene, pcr amplification and water;
(2) extraction, purification of samples DNA, measure its concentration, and the real-time quantitative PCR carrying out sample according to A, B and C tube reaction system of step (1) detects, and according to typical curve and amplification fluorescent signal, differentiate transgenation existence and Sudden Changing Rate.
In described step (1), the gene order of the GAG mutant plasmids standard substance of BRAF gene the 15th exon sudden change V600E is as shown in SEQID NO.17, and the gene order of the GAA mutant plasmids standard substance of BRAF gene the 15th exon sudden change V600E is as shown in SEQ IDNO.18;
The gene order of the plasmid standard of BRAF gene the 15th exon sudden change V600K is as shown in SEQ ID NO.19;
The gene order of BRAF gene wild plasmid standard substance is as shown in SEQ ID NO.16.
In step (1), the reaction conditions of PCR is: 95 DEG C of denaturation 10m, afterwards 95 DEG C of sex change 15s, 70-75 DEG C of PNA in conjunction with 15s, 60 DEG C annealing 60s, totally 50 circulations.
In described step (2), sample DNA comprises: the DNA extracted from ight soil, tissue, tissue juice, cell strain and blood.
In the present invention, in the detection that BRAF gene the 15th exon V600E suddenlys change, probe in detecting mutation type is the following two kinds:
GAG（E1）
GAA（E2）。
In detection for BRAF gene exon15V600K sudden change, probe in detecting mutation type is: AAG(K).
Table 2 sequence
Therefore, the present invention can solve following problems:
1, design PNA repressor sequences, the corresponding wild type sequence in mutation inhibiting site, to the interference of mutant nucleotide sequence specific amplification, namely eliminates the non-specific amplification of wild type sequence, improves specificity.
2, design PNA repressor sequences, suppress the cross interference between different mutant nucleotide sequence, namely eliminate the cross reaction between different mutant nucleotide sequence, improve specificity.
3, the PNA repressor sequences (as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.11 etc.) of design merger, makes the plan in same site detect the interference of Nucleotide from residue 3 kinds of Nucleotide.
4, the Taqman-MGB probe (comprise as shown in SEQ ID NO.6 sequence, comprise sequence as shown in SEQ ID NO.10) that annexs of design, makes the 3 kinds of mutated nucleotides being different from wild type sequence in same site all can be detected.
5, the assembling of forming a complete and comprehensive system for collecting real estate fees of PNA repressor sequences and Taqman-MGB probe.Merger PNA repressor sequences and merger Taqman-MGB probe and primer sets are loaded in a reaction system, realizes the specific detection to a certain mutational site.
6, the determination of reaction system.Comprise the consumption etc. of template, primer, PNA repressor sequences and Taqman-MGB probe and enzyme buffer liquid.
7, the determination of PCR response procedures.The determination of the temperature and time comprise sex change, check, increasing and amplification cycles number.
The present invention is based on peptide nucleic acid(PNA) in background technology as the feature that can complementary combine with DNA, itself and real-time quantitative PCR are detected and combines, be directed to BRAF of different nature sudden change and design fluorescently-labeled Taqman MGB probe respectively, be applied to BRAF gene exon15 abrupt climatic change, and the type of distinguishable BRAF gene exon15V600E and V600K sudden change.
The present invention is by the Taqman MGB probe (comprising: the merger probe that comprise as shown in SEQ ID NO.6 sequence) of design for V600E, design the Taqman MGB probe for V600K, design is for the merger Taqman MGB probe (comprising sequence as shown in SEQ ID NO.10) detecting V600E and V600K simultaneously, design the PNA repressor sequences for wild-type BRAF gene, design the PNA repressor sequences (comprise and annex sequence) etc. for other mutation types all possible beyond the mutation type intending detecting, namely by designing for the Taqman MGB probe of certain mutation type (comprising merging type) and the application form etc. that forms a complete set of of the corresponding PNA of checking sequence, thus realize the present invention.
The present invention adopts the real-time quantitative PCR method of associating peptide nucleic acid(PNA) (PNA) to detect BRAF gene the 15th exon V600E and V600K sudden change and Sudden Changing Rate.Because PNA covers the wild type sequence enclosing BRAF gene V600 site and the sequence intending other mutation types all possible beyond the mutation type of detection, and the mispairing of the PNA/DNA that the variation in arbitrary site causes in sequence, melting temp (Tm) is made to occur obviously to change, thus effectively can check the sequence of complete complementary, mismatch is increased, make plan detect sudden change effectively amplified and detect, wild-type and saltant type can be distinguished, and suppress other cross interference that it is caused of may suddenling change.Therefore, the present invention is that a kind of easy and simple to handle, susceptibility is high, specificity is high BRAF gene the 15th exon V600E and V600K suddenlys change Real_time quantitative detection method.
Beneficial effect of the present invention is as follows:
1) improve current PNA and close wild type sequence merely, design annexs PNA repressor sequences increases the ability that it closes other mutant nucleotide sequence, BRAF gene the 15th exon sudden change V600E and V600K is detected more accurate.
2) effectively by PNA repressor sequences and Taqman MGB probe connected applications, detection sensitivity is substantially increased.
Accompanying drawing explanation
Below in conjunction with accompanying drawing and embodiment, the present invention is further detailed explanation:
Fig. 1 is wild and the ABC pipe detected result of sudden change (3 kinds) plasmid reference sample, wherein, Fig. 1-A is V600E sudden change positive criteria product plasmid GAG, Fig. 1-B is V600E sudden change positive criteria product plasmid GAA, Fig. 1-C is V600K sudden change positive criteria product plasmid AAG, and Fig. 1-D is wild standard substance plasmid GTG;
Fig. 2 is the detected result of the continuous gradient dilute sample of V600E sudden change positive criteria product plasmid GAG, and wherein, Fig. 2-A is ABC pipe detected result; Fig. 2-B is typical curve, and wherein, the slope of typical curve is-3.951, Y-Inter(Y y-intercepts): 50.554, R
2(coefficient of determination of Linear Regression Model in One Unknown): 0.975, eff%(amplification efficiency): 79.11;
Fig. 3 is BRAF gene V600E sudden change and V600K abrupt climatic change pcr amplification figure in 8 routine Colorectal Carcinoma samples, and wherein, Fig. 3-A is V600E sudden change, and Fig. 3-B is V600K sudden change.
Embodiment
Describe the present invention below in conjunction with drawings and Examples.Following examples are to carry out Real_time quantitative detection at the plasmid containing BRAF gene V600E and V600K site mutation sequence, and the present invention is described in detail.Should be understood that this embodiment is only not used in for illustration of the present invention to limit the scope of the invention.
Embodiment 1 is for the detection of V600E and V600K mutant plasmid standard substance
V600E comprises two kinds of coding mutation sequence: GAG and GAA
Main agents is originated:
1.1 design PNA
PNA is synthesized by Korea S panagene and PNA Bio Inc. company of the U.S..
PNA-NBN-W:NH
2-TTGGTCTAGCTACA
nBNaAATC-COOH; Wherein, N represents A, C, G or T, and B represents C, G or T; All mix by 1:1 between described merger sequence.
PNA-HNN-K:NH
2-TTGGTCTAGCTACA
hNNaAATC-COOH; Wherein,
hrepresent A, C or T,
nrepresent A, C, G or T; All mix by 1:1 between described merger sequence.
PNA-BNN-E1E2:NH
2-TTGGTCTAGCTACA
bNNaAATC-COOH; Wherein,
brepresent C, G or T,
nrepresent A, C, G or T; All mix by 1:1 between described merger sequence.
1.2 design Tagman MGB probes
Tagman MGB probe is synthesized by prompt base (Shanghai) trade Co., Ltd in the English Weihe River.
Probe 1(Probe1 is wild): FAM(fluorescent mark)-TGGTCTAGCTACAGTGAA – MGB(minor groove binder)
Probe 2(Probe2 suddenlys change E1E2): FAM-TGGTCTAGCTACAGARAA – MGB
Probe 3(Probe3 suddenlys change K): FAM-TGGTCTAGCTACAAAGAA – MGB
1.3PCR primer
PCR primer is synthesized by prompt base (Shanghai) trade Co., Ltd in the English Weihe River.
Upstream primer sequence (Forward Primer):
5′-TCATGAAGACCTCACAGTAAAAATAGGT-3′（SEQ ID NO.1）
Downstream primer sequence (Reverse Primer):
5′-TGGGACCCACTCCATCGA-3′（SEQ ID NO.2）
1.4TaqMan Gene Expression Master Mix
Buy in AppliedBiosystems company of the U.S..
1.5BRAF gene wild-type and V600E and V600K mutant plasmids standard substance build
The wild-type of subordinate and mutant plasmids standard substance are synthesized by prompt base (Shanghai) trade Co., Ltd in the English Weihe River, and its gene order composition is respectively: wild:
CTGATAGGAAAATGAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCT
TCATGAAG ACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGTGAAATCTCGATGGAGTGGGTCC
CATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCT（SEQ ID NO.16）
Sudden change 1-1GAG(E):
CTGATAGGAAAATGAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGAGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCT（SEQ ID NO.17）
Sudden change 1-2GAA(E):
CTGATAGGAAAATGAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCT
TCATGAAG ACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGAAAAATCTCGATGGAGTGGGTCC
CATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCT（SEQ ID NO.18）
Sudden change 2AAG(K):
CTGATAGGAAAATGAGATCTACTGTTTTCCTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAAAGAAATCTCGATGGAGTGGGTCCCATCAGTTTGAACAGTTGTCTGGATCCATTTTGTGGATGGTAAGAATTGAGGCTATTTTTCCACTGATTAAATTTTTGGCCCT（SEQ ID NO.19）
Be 10 by above-mentioned 4 kinds of plasmid standard doubling dilutions
6, 10
5, 10
4, 10
3, 10
2, 10
1, 0 copy, each concentration gradient all containing normal human subject genomic dna 125ng, with this production standard curve.Meanwhile, by 10 of 4 kinds of plasmid standards
5copy number DNA is as reference sample.
1.6 Real_time quantitative detection reaction system and reaction conditionss
7500 type real-time PCRs of instrument selection ABI company.Sample detection carries out three tube reactions simultaneously.PCR reaction conditions: 95 DEG C of denaturation 10m, afterwards 95 DEG C of sex change 15s, 70-75 DEG C of PNA in conjunction with 15s, 60 DEG C annealing 60s, totally 50 circulations.
A manages: V600E detects, and adds PNA, is namely called A-PNA+
B manages: V600K detects, and adds PNA, is namely called B-PNA+
C manages: total V600 detects, and does not add PNA, is namely called C-PNA-
A tube reaction system is as shown in table 3.
Table 3A tube reaction system
B tube reaction system is as shown in table 4.
Table 4B tube reaction system
C tube reaction system is as shown in table 5.
Table 5C tube reaction system
The Specification Curve of Increasing of detection by quantitative:
The DNA standard substance template continuous gradient dilution of sudden change 1-1 or sudden change 1-2 and sudden change 2 is 10
6, 10
5, 10
4, 10
3, 10
2, 10
1, 0 copy, carry out corresponding A pipe or B pipe and detect, production standard curve.
1.7 detect the specificity of this detection method according to standard substance
According to the specific outcome that can obtain this detection method to the reference sample inspection of plasmid standard.
In the ABC pipe detected result of wild and sudden change (3 kinds) plasmid reference sample as shown in Figure 1, Fig. 1-A shows V600E sudden change positive criteria product plasmid GAG has remarkable detection signal (less CT value) at A pipe, at B pipe without effective detection signal, there is remarkable detection signal (less CT value) at C pipe; Be indicated as V600E sudden change.Fig. 1-B shows shown in the similar Fig. 1 of result-A of V600E sudden change positive criteria product plasmid GAA; Be indicated as V600E sudden change equally.Fig. 1-C display V600K sudden change positive criteria product plasmid AAG without detection signal at A pipe, has remarkable detection signal (less CT value) at B pipe, has remarkable detection signal (less CT value) at C pipe; Be indicated as V600K sudden change.Fig. 1-D shows wild standard substance plasmid GTG at A and B pipe without detection signal, has remarkable detection signal (less CT value) at C pipe; Being indicated as V600 site and there is not sudden change, is wild-type.
Conclusion, this detection method specific detection can go out V600E sudden change and V600K sudden change respectively, not by cross interference that is wild and other mutation type in specificity detects.
1.8 judge susceptibility and the quantivative approach of this detection method according to typical curve
According to 10 to plasmid standard
6, 10
5, 10
4, 10
3, 10
2, 10
1, 0 copy the detection of continuous gradient dilute sample, the sensitivity that the method detects can be obtained.The detected result of the continuous gradient dilute sample of V600E sudden change positive criteria product plasmid GAG as shown in Figure 3, show that sample CT value that the method detects can reference standard curve (as shown in Figure 2), converse the copy number quantitative values (wherein, V600E suddenly change the relation of the copy number of continuous gradient dilute sample of positive criteria product plasmid GAG and CT value as shown in table 6) of mutator gene in sample.In like manner.GAA and AAG also can obtain equifinality.
The relation of table 6 copy number and CT value
| Copy number | CT value (A or B pipe) |
| 2000 | 35.47 |
| 1000 | 36.75 |
| 500 | 37.67 |
| 250 | 38.09 |
| 125 | 40.09 |
| 63 | 41.01 |
| 0 | 40.91 |
In addition, involved in the present embodiment reagent comprises plasmid, probe, PNA sequence, primer, PCR reaction solution etc. can be assembled into the test kit detecting V600E and V600K sudden change.
The detection that embodiment 2 is suddenlyd change for V600E and V600K of Colorectal Carcinoma sample
2.1 specimen collections:
Collect fresh Colorectal Carcinoma sample cancer 8 example (diagnosis of cancer is all foundation with Clinicopathologic Diagnosis, comprises pathological diagnosis) of excision, organize about 1mg.
2.2DNA method for extracting:
Select commercially available Qiangen company genome DNA extraction test kit, by specification carries out genome DNA extraction, with 100 μ lTE dissolving DNAs, is sub-packed in-80 DEG C of preservations.
2.3DNA concentration determination
The long ultraviolet/visible light scanning spectrophotometer of application NanoDrop ND-1000 all-wave measures concentration.
2.4 samples carry out V600E and V600K abrupt climatic change
Sample thief DNA125ng, carries out ABC tri-pipe according to the method 1.6 in embodiment 1 and detects.
2.5 interpretation of result
With reference to 1.7 specificity analysis methods in embodiment 1, see Fig. 3, A pipe V600E abrupt climatic change, result finds that 1 example is positive, B pipe V600K does not find positive amplification.
Result judges have 1 routine BRAF V600E to suddenly change in 8 routine Colorectal Carcinoma samples, and all the other are wild-type.
Claims (21)
1. a pair for detect BRAF gene the 15th exon sudden change V600E and V600K primer pair, it is characterized in that, comprising:
1) upstream sequence as shown in SEQ ID NO.1 and the downstream sequence as shown in SEQ ID NO.2; Or
2) at least identical with the upstream sequence 80% shown in SEQ ID NO.1 sequence and at least identical with the downstream sequence 80% shown in SEQ ID NO.2 sequence.
2. for detecting a probe for wild-type BRAF gene, it is characterized in that, comprising: the probe comprising sequence shown in SEQ ID NO.3.
3. probe as claimed in claim 2, is characterized in that: the two ends of described probe are also marked with fluorescent reporter group and fluorescent quenching group respectively;
Wherein, fluorescent reporter group comprises: FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA;
Fluorescent quenching group comprises: MGB or TAMRA.
4. one kind for detect BRAF gene the 15th exon sudden change V600E probe, it is characterized in that, comprising: comprise the probe of sequence shown in SEQ ID NO.4, comprise the probe of sequence shown in SEQ ID NO.5 or comprise the probe of sequence shown in SEQ ID NO.6.
5. probe as claimed in claim 4, is characterized in that: the two ends of described probe are also marked with fluorescent reporter group and fluorescent quenching group respectively;
Wherein, fluorescent reporter group comprises: FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA;
Fluorescent quenching group comprises: MGB or TAMRA.
6., for detecting a PNA repressor sequences of BRAF gene the 15th exon sudden change V600E, it is characterized in that, comprise: the PNA repressor sequences shown in SEQ IDNO.7 or the PNA repressor sequences shown in SEQ ID NO.8;
SEQ ID NO.7:TTGGTCTAGCTACANBNAAATC; Wherein, N represents A, C, G or T, and B represents C, G or T;
SEQ ID NO.8:TTGGTCTAGCTACAHNNAAATC; Wherein, H represents A, C or T, and N represents A, C, G or T.
7. PNA repressor sequences as claimed in claim 6, is characterized in that: the NBN in described SEQ ID NO.7 is selected from following combination:
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, CCT, CCC, CCG, CGA, CGT, CGC, CGG, CTA, CTT, CTC or CTG;
HNN in SEQ ID NO.8 is selected from following combination:
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 repressor sequences as claimed in claim 6, it is characterized in that: the described PNA repressor sequences for detecting BRAF gene the 15th exon sudden change V600E, comprising: the PNA repressor sequences shown in the PNA repressor sequences shown in SEQ ID NO.7 and SEQ ID NO.8;
Wherein, the NBN in SEQ ID NO.7 comprises: GTG, ATG and AGG;
HNN in SEQ ID NO.8 comprises: AAG, ATG and AGG.
9. PNA repressor sequences as claimed in claim 6, it is characterized in that: the described PNA repressor sequences for detecting BRAF gene the 15th exon sudden change V600E, comprising: the PNA repressor sequences as shown in SEQ ID NO.12 and the PNA repressor sequences as shown in SEQ ID NO.15.
10., for detecting a probe of BRAF gene the 15th exon sudden change V600K, it is characterized in that, comprise: the probe comprising sequence shown in SEQ ID NO.9.
11. probes as claimed in claim 10, is characterized in that: the two ends of described probe are also marked with fluorescent reporter group and fluorescent quenching group respectively;
Wherein, fluorescent reporter group comprises: FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA;
Fluorescent quenching group comprises: MGB or TAMRA.
12. 1 kinds, for detecting the merger probe of BRAF gene the 15th exon sudden change V600E and V600K, is characterized in that, comprising: the probe comprising sequence shown in SEQ ID NO.10;
SEQ ID NO.10:TGGTCTAGCTACARARAA; Wherein, R is A or G.
13. probes as claimed in claim 12, is characterized in that: the two ends of described probe are also marked with fluorescent reporter group and fluorescent quenching group respectively;
Wherein, fluorescent reporter group comprises: FAM, ViC, NED, Cy3, Cy5, JOE, TEXASRED or TAMRA;
Fluorescent quenching group comprises: MGB or TAMRA.
14. 1 kinds, for detecting the PNA repressor sequences of BRAF gene the 15th exon sudden change V600K, is characterized in that, comprising: the PNA repressor sequences shown in SEQID NO.11;
SEQ ID NO.11:TTGGTCTAGCTACABNNAAATC; Wherein, B represents C, G or T, and N represents A, C, G or T.
15. PNA repressor sequences as claimed in claim 14, is characterized in that: the BNN in described SEQ ID NO.11 is selected from following combination:
CAA, CAT, CAC, CAG, CTA, CTT, CTC, CTG, CCA, CCT, 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, TCC, TCG, TGA, TGT, TGC or TGG.
16. PNA repressor sequences as claimed in claim 14, it is characterized in that: described PNA repressor sequences, comprising: the PNA repressor sequences as shown in SEQ ID NO.12, the PNA repressor sequences as shown in SEQ ID NO.13 and the PNA repressor sequences as shown in SEQ ID NO.14.
17. 1 kinds, for detecting the test kit of BRAF gene the 15th exon sudden change V600E and V600K, is characterized in that, comprising:
Primer pair according to claim 1, probe for detecting wild-type BRAF gene according to claim 2, according to claim 4 for detect BRAF gene the 15th exon sudden change V600E probe or according to claim 12 for detect BRAF gene the 15th exon sudden change V600E and V600K merger probe, PNA repressor sequences for detecting BRAF gene the 15th exon sudden change V600E according to claim 6, according to claim 10 for detect BRAF gene the 15th exon sudden change V600K probe or according to claim 12 for detect BRAF gene the 15th exon sudden change V600E and V600K merger probe, and the PNA repressor sequences for detecting BRAF gene the 15th exon sudden change V600K according to claim 14.
18. test kits as claimed in claim 17, is characterized in that: described test kit, comprising:
1) pair of primers pair, its sequence is the such as upstream sequence shown in SEQ ID NO.1 and the downstream sequence as shown in SEQ ID NO.2;
2) probe of sequence shown in SEQ ID NO.3 is comprised;
3) for detect BRAF gene the 15th exon sudden change V600E probe, its for comprise sequence shown in SEQ ID NO.4 probe, comprise the probe of sequence shown in SEQ ID NO.5 or comprise the probe of sequence shown in SEQ ID NO.6; Or
For detecting the merger probe of BRAF gene the 15th exon sudden change V600E and V600K;
4) for detecting the PNA repressor sequences of BRAF gene the 15th exon sudden change V600E, it is the PNA repressor sequences shown in the PNA repressor sequences shown in SEQ ID NO.7 and SEQ ID NO.8, or the combination be made up of the PNA repressor sequences shown in SEQ ID NO.12 and the PNA repressor sequences shown in SEQ IDNO.15;
5) for detecting the probe of BRAF gene the 15th exon sudden change V600K, it is for comprising the probe of sequence shown in SEQ ID NO.9 or the merger probe for detecting BRAF gene the 15th exon sudden change V600E and V600K;
6) for detecting the PNA repressor sequences of BRAF gene the 15th exon sudden change V600K, it is the PNA repressor sequences shown in SEQ ID NO.11 or the combination that is made up of the PNA repressor sequences such as shown in SEQ ID NO.12, the PNA repressor sequences as shown in SEQ ID NO.13 and the PNA repressor sequences as shown in SEQID NO.14.
19. test kits as claimed in claim 17, it is characterized in that: described test kit, also comprises: BRAF gene wild plasmid standard substance, BRAF gene V600E mutant plasmids standard substance, BRAF gene V600K mutant plasmids standard substance, PCR damping fluid, dNTPs and archaeal dna polymerase.
20. 1 kinds of methods utilizing the test kit described in claim 17 to carry out Real_time quantitative detection BRAF gene the 15th exon sudden change V600E and V600K, is characterized in that, comprise step:
(1) carry out following A, B and C tube reaction simultaneously, the real-time quantitative PCR that the plasmid standard of known mutations amount carries out suddenling change about V600E and V600K of BRAF gene is detected, draws typical curve;
Wherein, the reaction system of A pipe comprises: the GAA mutant plasmids standard substance of BRAF gene the 15th exon sudden change GAG mutant plasmids standard substance of V600E or BRAF gene the 15th exon sudden change V600E, above-mentioned primer pair, for detect BRAF gene the 15th exon sudden change V600E probe or for detect BRAF gene the 15th exon sudden change V600E and V600K merger probe, for detecting enzyme buffer liquid needed for the PNA repressor sequences of BRAF gene the 15th exon sudden change V600E, pcr amplification and water;
The reaction system of B pipe comprises: BRAF gene the 15th exon sudden change plasmid standard of V600K, above-mentioned primer pair, for detect BRAF gene the 15th exon sudden change V600K probe or for detect BRAF gene the 15th exon sudden change V600E and V600K merger probe, for detecting enzyme buffer liquid needed for the PNA repressor sequences of BRAF gene the 15th exon sudden change V600K, pcr amplification and water;
The reaction system of C pipe comprises: BRAF gene wild plasmid standard substance, above-mentioned primer pair, for detecting enzyme buffer liquid needed for the probe of wild-type BRAF gene, pcr amplification and water;
(2) extraction, purification of samples DNA, measure its concentration, and the real-time quantitative PCR carrying out sample according to A, B and C tube reaction system of step (1) detects, and according to typical curve and amplification fluorescent signal, differentiate transgenation existence and Sudden Changing Rate.
21. methods as claimed in claim 20, it is characterized in that: in described step (1), the gene order of the GAG mutant plasmids standard substance of BRAF gene the 15th exon sudden change V600E is as shown in SEQ ID NO.17, and the gene order of the GAA mutant plasmids standard substance of BRAF gene the 15th exon sudden change V600E is as shown in SEQ ID NO.18;
The gene order of the plasmid standard of BRAF gene the 15th exon sudden change V600K is as shown in SEQ ID NO.19;
The gene order of BRAF gene wild plasmid standard substance is as shown in SEQ ID NO.16;
In step (1), the reaction conditions of PCR is: 95 DEG C of denaturation 10m, afterwards 95 DEG C of sex change 15s, 70-75 DEG C of PNA in conjunction with 15s, 60 DEG C annealing 60s, totally 50 circulations;
In step (2), sample DNA comprises: the DNA extracted from ight soil, tissue, tissue juice, cell strain and blood.
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Application publication date: 20150923 |