CN105039514A - Detection kit for human BRAF gene V600E mutation - Google Patents
Detection kit for human BRAF gene V600E mutation Download PDFInfo
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Abstract
The invention provides a detection kit and a detection method for human BRAF gene V600E mutation. The detection kit comprises a first reagent pack and a second reagent pack, wherein each of the first reagent pack and the second reagent pack comprises PCR buffer solution, dNTP, MgCl2, a specific probe, a locked nucleic acid (LNA) primer, an interior label system, HotStart Taq enzyme and UNG enzyme; the first reagent pack comprises a primer pair SEQ ID NO:1 and SEQ ID NO:2, and a probe SEQ ID NO:3; the second reagent pack comprises a primer pair SEQ ID NO:1 and SEQ ID NO:4, and a probe SEQ ID NO:5; and the SEQ ID NO:4 is LNA primer. The detection kit and the detection method can be used for detecting human BRAF gene V600E mutation, and has the advantages of strong specificity, high sensitivity, simplicity and rapidness in operation, high flux, safety, objective result determination and the like.
Description
Technical field
The present invention relates to a kind of test kit of mankind BRAF gene V600E abrupt climatic change, particularly relate to a kind of test kit fast and effeciently detecting the BRAF gene V600E sudden change of a small amount of sample.
Background technology
BRAF gene, full v-Raf murine sarcoma virus proto-oncogene homologue B1 (v-RafmurinesarcomaviraloncogenehomologB1) by name, found in induction fowl primary cell propagation and NIH3T3 transformation process by IKawa etc. and cloned confirmation for 1988, with ARAF, CRAF belongs to RAF gene family, be positioned at human chromosome 7q34, size is about 190kb, its coding region with function is by 2510 pairs of based compositions, serine threonine protein kinase in coding MAPK path, signal is transduceed to MEK1/2 from RAS by this enzyme, thus various biological event in participation regulating cell.
BRAF sudden change occurs in nearly 8% human tumor, mainly betides in colorectal carcinoma, melanoma and thyroid papillary carcinoma.According to statistics, about 15% colorectal cancer patients in body cell BRAF gene mutation.The sudden change of 11% is positioned at the glycine ring on exons 11; The sudden change of 89% betides the active region on exons 15, and wherein about 92% is positioned on the 1799th Nucleotide (T sports A), and the α-amino-isovaleric acid causing it to encode replaces (V600E) by L-glutamic acid.There is research to point out, for KRAS gene wild-type there is the colorectal cancer patients of BRAFV600E sudden change simultaneously, use anti-egfr antibodies targeted therapy invalid.2011 " NCCN colorectal carcinoma clinical practice guideline " suggestion reply KRAS gene wild-type patient detects its BRAF gene type further.
Table 1:BRAF gene V600E sudden change
Detection method at present for transgenation is a lot, as direct sequencing, and Manganic pyrophosphate complex initiation method, high resolving power solubility curve detection method (HighResolutionMeltingAnalysis, HRM), fluorescence quantitative PCR method etc.Wherein most common methods is sequencing, and the method expense is lower, but operates length consuming time and sensitivity is low; High resolving power solubility curve method is more special to equipment requirements, there is certain difficulty at clinical expansion; Conventional fluorescent quantitative PCR method is applied comparatively extensive clinically, but the method detection deletion mutantion effect is poor, and accuracy is not high.Therefore, need to set up a kind of method detecting mankind BRAF gene V600E abrupt climatic change fast and effectively.
Summary of the invention
The object of the invention is, overcomes deficiency of the prior art, provides the detection kit that a kind of mankind BRAF gene V600E suddenlys change, and solves in existing genetic polymorphism detection technology with this, the problem that when sample size is few, genetic polymorphism detection effect is undesirable.
The invention provides detection kit and the detection method of a kind of mankind BRAF gene V600E sudden change.This test kit comprises the first pack and the second pack, described first pack and each self-contained PCR damping fluid of the second pack, dNTP, MgCl
2specific probe, lock nucleic acid primer, interior mark system, HotStartTaq enzyme, UNG enzyme, wherein the first pack comprises primer pair SEQIDNO:1 and SEQIDNO:2, probe SEQIDNO:3, second pack comprises primer pair SEQIDNO:1 and SEQIDNO:4, probe SEQIDNO:5, and wherein SEQIDNO:4 is lock nucleic acid primer.
In some embodiments of the present invention, the 5 ' end of probe SEQIDNO:3, SEQIDNO:5 is connected with fluorophor, and 3 ' end is connected with quenching group.
In some embodiments of the present invention, described interior mark system comprises interior label primer and interior mark probe, and described interior label primer sequence is SEQIDNO:6 and SEQIDNO:7, and described interior mark probe sequence is SEQIDNO:8.
In some embodiments of the present invention, described interior mark probe 5 ' is terminal modified VIC, and 3 ' terminal modifiedly has TAMRA.
In some embodiments of the present invention, in the first pack and the second pack all containing ROX reference corrected.
In some embodiments of the present invention, described detection kit contains positive control solution and blank liquid, and described positive control solution contains V600E mutant plasmid DNA, and described blank is Tris-HCl damping fluid.
Mankind BRAF gene V600E mutation detection kit of the present invention can in simple and quick detection gene pleiomorphism, described detection comprises the following steps: (1) is designed and screened primer, the probe containing the Quality Control of mankind's BRAF gene, detection probes containing BRAF gene V600E sudden change, ARMS primer, universal primer; (2) testing sample genomic dna is obtained; (3) detection kit according to any one of claim 1-5 is got, described genomic dna is successively got identical amount to add in described first pack and described second pack and to carry out PCR reaction simultaneously, the difference △ Ct of the Ct value obtained respectively according to two PCR reaction systems judges whether to have corresponding transgenation.
Test kit of the present invention has the following advantages: 1. the transgenation situation that accurately can detect single sample; 2. accurately can detect the BRAF gene mutation of in 10ng genomic dna sample 0.1%; 3. use fluorescent quantitative PCR technique to detect, testing process is stopped pipe reaction, significantly reduces pollution, and adds UNG enzyme anti-pollution system, guarantee that real result is credible; 5. in two reaction tubess, detect same sample, easy and simple to handle, detected result interpretation method can be completed in 90 minutes simply objective, be convenient to analyze.
Present invention also offers and utilize detection kit of the present invention to carry out the method detected, the method comprises the following steps:
(1) testing sample genomic dna is obtained;
(2) detection kit of the present invention is got, the genomic dna of above-mentioned acquisition is successively got identical amount to add in described first pack and described second pack and to carry out PCR reaction simultaneously, the difference △ Ct of the Ct value obtained respectively according to two PCR reaction systems judges whether to have corresponding transgenation.
The present invention adopts fluorescence quantifying PCR method, specific amplification detection is carried out to BRAF gene V600E mutant nucleotide sequence, lock nucleic acid (LNA) technology is introduced in augmentation detection system, lock nucleic acid is a kind of class oligonucleotide through modifying, the 2'-O of β-D-RIBOSE in structure, 4'-C position forms rigid structure by shrink effect, have and DNA, the binding ability that RNA is powerful, we utilize lock nucleic acid design abrupt climatic change primer sequence, make it the specific combination with mutant DNA template in testing sample, affinity is higher than corresponding oligonucleotide a lot, the melting temperature(Tm) forming hybridization chain is higher.And lock nucleic acid primer and not complementary wild-type DNA and other mutant DNAs of similar V600 (V600K, V600D, V600M, V600R) hybridizing avidity declines more, and melting temperature(Tm) is low, causes amplification efficiency well below mutagenesis template, again through the screening of V600E specific probe, make the combination fully suppressing wild-type DNA profiling and probe, realize wild-type template zero-signal, can ensure that saltant type template amplification has highly sensitive simultaneously.Detection method of the present invention is simply effective, and detection sensitivity is high, fast simple to operate, and result interpretation is simply objective, is a kind of method effectively detecting mankind BRAF gene V600E sudden change fast.
Accompanying drawing explanation
Fig. 1 is a negative result figure of an embodiment of detection kit of the present invention;
Fig. 2 is a positive test symbol figure of an embodiment of detection kit of the present invention;
Fig. 3 is another negative result figure of an embodiment of detection kit of the present invention;
Fig. 4 is another positive test symbol figure of an embodiment of detection kit of the present invention.
Embodiment
In order to make the technical problem to be solved in the present invention, technical scheme and beneficial effect clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiments described herein only in order to explain the present invention, do not limit the present invention.
One embodiment of the invention provide a kind of test kit, comprise PCR damping fluid, dNTP, MgCl
2, specific probe, lock nucleic acid primer, interior mark system, HotStartTaq enzyme, UNG enzyme, be divided into the first pack and the second pack, wherein the first pack comprises primer pair SEQIDNO:1 and SEQIDNO:2, probe SEQIDNO:3, the second pack comprises primer pair SEQIDNO:1 and SEQIDNO:4, probe SEQIDNO:5, SEQIDNO:3, SEQIDNO:5 are MGB probe sequence, its 5 ' end is connected with fluorophor, and 3 ' end is connected with quenching group NFQ, and SEQIDNO:4 is lock nucleic acid primer.
In some specific embodiments, the first pack and the second pack can form PCR reaction system with other components, or self include PCR and react necessary component to carry out fluorescence quantitative PCR detection.When the first pack and the second pack are separately as PCR reaction system, first pack is except comprising primer pair SEQIDNO:1 and SEQIDNO:2, outside probe SEQIDNO:3, also comprise PCR damping fluid, Taq enzyme, and by the supplementary volume that adds water, wherein contain magnesium ion and dNTP in this PCR damping fluid.Second pack contains general primer SEQIDNO:1 and lock nucleic acid primer SEQIDNO:4, probe SEQIDNO:5, also comprise PCR damping fluid, Taq enzyme, and supplement volume by water, wherein contain magnesium ion and dNTP in PCR damping fluid, in the second pack, also comprise interior mark system interior label primer sequence is SEQIDNO:6 and SEQIDNO:7, described interior mark probe sequence is SEQIDNO:8, respectively testing sample DNA can be added in above-mentioned first pack and the second pack when detecting and carry out PCR detection, compare difference according to the Ct value of the detected result of the PCR reaction system respectively containing above-mentioned first pack and the second pack and determine whether detected result is the positive.
In some specific embodiments, above-mentioned primer, probe and blocking-up nucleotide sequence are respectively:
Forward primer SEQIDNO:15 '-GAATTATAGAAATTAGATCTCTTACC-3 '
Reverse primer SEQIDNO:25 '-ACAAAATGGATCCAGACAACTGT-3 '
Probe SEQIDNO:35 '-FAM-TGATAGGAAAATGAGATC-3 '-NFQ-MGB,
Lock nucleic acid primer SEQIDNO:45 '-ACGATGGGACCCACTCCATCGAGATTT+T-3 '
Probe SEQIDNO:55 '-FAM-TTGGTCTAGCTACAGAGA-3 '-NFQ-MGB
Interior mark forward primer SEQIDNO:65 '-CGCGAACTCACCCGT-3 '
Interior mark reverse primer SEQIDNO:75 '-CACTAGGCGCTCACTGT-3 '
Interior mark probe SEQIDNO:85 '-VIC-CACCTTCCCCATGGTGTCT-3 '-NFQ-MGB
In some specific embodiments, above-mentioned probe sequence 5 ' holds the fluorophor connected can be FAM or VIC fluorophor, can select other suitable fluorophor, such as ROX, HEX in addition.The quenching group NFQ that this probe sequence 3 ' end connects can be quenching group well known to those skilled in the art.The fluorophor that probe 5 ' is held and the 3 ' quenching group held close to each other time, fluorescent reporter group can not send fluorescence, but along with the carrying out of pcr amplification reaction, 5 ' the fluorophor held splits away off along with the hydrolysis of probe, thus can fluorescence be sent, quantitative analysis can be carried out to unknown template by the accumulation detecting fluorescent signal.3 ' end of this probe is connected with MGB (MinorGrooveBinder) group, and MGB molecular juction is incorporated into DNA spiral ditch, is improved the test effect of hybridization by stable MGB probe/template association.This MGB group can by the Tm value raising about 10 DEG C of probe, and therefore for same Tm value, MGB probe can obtain shorter than general T aqMan probe design, specificity is stronger.
Detection kit provided by the invention is easy and simple to handle, and sense cycle is short, achieves the rapid detection for sample.
One embodiment of the invention also provide a kind of detection method of suddenling change for mankind BRAF gene V600E, and this detection method utilizes detection kit provided by the invention to carry out, and this detection method comprises the following steps:
(1) testing sample genomic dna is obtained;
(2) genomic dna of above-mentioned acquisition successively being got same amount adds in the PCR reaction system containing the first pack in detection kit of the present invention and the PCR reaction system containing the second pack in detection kit of the present invention respectively, and make these two reaction systems carry out PCR reaction simultaneously, two PCR reaction is carried out independently of one another, and the difference △ Ct of the Ct value obtained respectively according to two PCR reaction systems judges whether to have corresponding transgenation.
Ct value corresponding to such as, PCR reaction system in some specific embodiments, containing the first pack is Quality Control value Ct
0, and be Ct containing the Ct value that the PCR reaction system of the second pack is corresponding
1, △ Ct=Ct
1-Ct
0.First according to Quality Control value Ct
0as the standard whether applied sample amount is suitable, Ct
0≤ 32 applied sample amounts are suitable, and detected result is effective; Ct
0>32 then applied sample amount is on the low side, need again detect, and detects after can suitably improving applied sample amount when again detecting again.Can judge the result of BRAF gene V600E sudden change while applied sample amount is judged: △ Ct value >=9 (the PCR reaction system comprised containing the second pack carry out PCR reaction after without the situation of Ct value) then sample suddenly change without BRAF gene V600E or abundance of suddenling change lower than this test kit abrupt climatic change lower limit, as shown in figs. 1 and 3; There is BRAF gene V600E sudden change in △ Ct<9 then sample, as shown in Figures 2 and 4.
In some specific embodiments, the Monitoring lower-cut of detection method provided by the invention is 10ng0.1%, and abundance of namely suddenling change can be detected higher than the sample of 0.1%.
In some embodiments of the present invention, can also by raise △ Ct value detect suddenly change abundance lower than 0.1% sample.
In some specific embodiments, the above-mentioned reaction volume containing the PCR reaction system of the first pack can be any volume being applicable to quantitative fluorescent PCR reaction.In the preferred embodiments of the invention, the above-mentioned reaction volume containing the PCR reaction system of the first pack is 25 μ l, can according to following proportions:
Containing dNTP in above-mentioned PCR damping fluid, magnesium ion etc. carry out PCR and react necessary component.
In some specific embodiments, the above-mentioned reaction volume containing the PCR reaction system of the second pack can be any volume being applicable to quantitative fluorescent PCR reaction.In some specific embodiments, the above-mentioned reaction volume containing the PCR reaction system of the second pack is 25 μ l, can according to following proportions:
Containing dNTP in above-mentioned PCR damping fluid, magnesium ion etc. carry out PCR and react necessary component.
In some specific embodiments, same component (except water) the above-mentioned PCR reaction system containing the first pack with containing the PCR reaction system of the second pack in content identical, namely identical with containing each component in the PCR reaction system of the first pack containing each component concentration in the PCR reaction system of the second pack, i.e. primer pair SEQIDNO:1 and SEQIDNO:2 in the first pack reaction system, probe SEQIDNO:3, PCR damping fluid, primer pair SEQIDNO:1 and SEQIDNO:4 in Taq enzyme composition and content and the second pack reaction system, probe SEQIDNO:5, PCR damping fluid, Taq enzyme composition and content all identical.
In some specific embodiments, when carrying out BRAF gene V600E abrupt climatic change, above-mentioned two kinds of PCR reaction systems are carried out under the same reaction conditions.In some specific embodiments, the PCR reaction conditions of above-mentioned two kinds of reaction systems is:
95℃5～10min；
95 DEG C of 15 ~ 30s, 60 DEG C of 45 ~ 60s, 40 ~ 45 circulations;
After above-mentioned each circulation, collect fluorescent signal, this process is completed automatically by reaction kit.
Some embodiments of the present invention have employed fluorescence quantifying PCR method, specific amplification detection is carried out to BRAF gene V600E Sudden change region sequence, utilize lock nucleic acid to add TaqMan probe two knowledge method for distinguishing simultaneously, realize the zero-signal of wild-type background and the specific amplification of deletion mutantion.Detection method of the present invention is simply effective, and detection sensitivity is high, fast simple to operate, and result interpretation is simply objective, is the method for a kind of effective detection mankind BRAF gene V600E sudden change.
Below by way of specific embodiment, the present invention will be described in detail.
Embodiment 1
Preparation mankind BRAF gene V600E mutation detection kit, specifically comprises the following steps:
1. synthetic primer and probe sequence
Synthetic primer sequence SEQIDNO:1, SEQIDNO:2, interior label primer sequence SEQIDNO:6 and SEQIDNO:7; Synthesis specific probe sequence SEQIDNO:3 and SEQIDNO:5, and at 5 ' end flag F AM fluorophor, 3 ' end is marked with quenching group NFQ and MGB modification group, mark probe sequence SEQIDNO:8 in synthesis, and at 5 ' end mark VIC fluorophor, 3 ' end is marked with quenching group NFQ and MGB modification group.
The mother liquor above-mentioned primer sequence being mixed with respectively 100 μMs stores, and the mother liquor above-mentioned probe sequence being mixed with respectively 100 μMs stores.
2. synthesis lock nucleotide sequence
Synthesis lock nucleic acid primer sequence SEQIDNO:4, and carry out 2' and 4' carbon connection modification at 3 ' end.
The mother liquor that lock nucleic acid primer makes 100 μMs is stored.
3. the preparation of quantitative fluorescent PCR reaction system
The Quality Control detection reaction system of preparation containing the first pack and the abrupt climatic change reaction system containing the second pack respectively, each component is as shown in table 2 below:
The composition of table 2 Quality Control detection reaction system and abrupt climatic change reaction system
Above-mentioned PCR damping fluid, for buy from market, wherein reacts necessary component containing the PCR such as dNTP, magnesium ion.
Embodiment 2
With BRAF gene V600E mutation detection kit prepared by embodiment 1, testing sample is detected.
The paraffin-embedded tissue section that 50 routine Clinicopathologic Diagnosis are thyroid cancer patients is collected in the present embodiment, and therefrom extract genomic dna, detect in testing sample whether there is BRAF gene V600E sudden change with the BRAF gene V600E mutation detection kit obtained in embodiment 1, adopt the method for tradition order-checking to verify, concrete operation step is simultaneously:
1. the extraction of sample gene group DNA
DNA extraction kit (QIAampDNAFFPETissueKit, CatNo.56404) is used to extract the genomic dna of the tissue sample of above-mentioned patients with lung cancer.DNA extraction method is carried out with reference to specification sheets, operation steps is summarized as follows: be placed in the centrifuge tube of 1.5ml by the paraffin-embedded tissue (≤25mg) of clinical acquisitions, add the dimethylbenzene of 1200 μ l, the centrifugal 5min of violent vortex 10s, 12000rpm room temperature.Abandon supernatant, note not outwelling precipitation.Add 1200 μ l dehydrated alcohols, to remove residual dimethylbenzene, vortex gently.The centrifugal 5min of 12000rpm room temperature.Abandon supernatant, again add 1200 μ l dehydrated alcohols, vortex gently.The centrifugal 5min of 12000rpm room temperature.Abandon supernatant, open centrifuge tube, hatch 10-15min at 37 DEG C, until ethanol evaporates completely.Add 180 μ lbufferATL.Add 20 μ l Proteinase Ks, thorough vortex, hatch about 2h for 56 DEG C, until this tissue dissolves (in the process of hatching can once in a while vortex) completely, vortex 15s afterwards, adds 200 μ lbufferAL, 200 μ l dehydrated alcohols are added, thoroughly vortex concussion again after mixing after vortex.Join on centrifugal column by last mixture, the centrifugal 1min of 8000rpm, abandons waste liquid.Add 500 μ lbufferAW1, the centrifugal 1min of 8000rpm, abandons waste liquid.Add 500 μ lbufferAW2, the centrifugal 3min of 12000rpm, abandon the centrifugal 1min of 12000rpm after waste liquid, be placed on by centrifugal column in new 1.5ml centrifuge tube, add 50 ~ 200 μ lbufferAE, room temperature places the centrifugal 1min of 1min, 12000rpm.Get 2 μ l gained solution and survey OD values to determine DNA concentration, then sample DNA is diluted to 10ng/ μ l, get 5 μ l respectively and to be added in test kit obtained in embodiment 1 and the PCR carrying out next step reacts.
2. the fluorescence quantitative PCR detection of sample
DNA sample after dilution in step 1 is got successively in the Quality Control detection reaction system and abrupt climatic change reaction system that 5 μ l add the test kit of embodiment 1 respectively, two kinds of reaction system cumulative volumes are made to be 25 μ l, and put into quantitative real time PCR Instrument, carry out amplified reaction by after the PCR of setting response procedures as follows:
37℃10min；
95℃5min；
95 DEG C of 15s, 60 DEG C of 1min, 40 circulations; FAM fluorescent signal is collected after each circulation.
3. the Analysis of test results of sample
The present invention utilizes the amplification blocking nucleotide sequence and suppress wild-type DNA profiling sequence, and detects the amplification that deletion mutantion DNA profiling occurs, by the Quality Control value Ct of Quality Control detection reaction system by collecting FAM fluorescent signal
0whether judgement sample is effective, by abrupt climatic change reaction system abrupt climatic change value Ct
1with Quality Control value Ct
0difference △ Ct judge whether occur BRAF gene V600E sudden change.
Concrete result interpretation standard is as follows:
Quality Control value Ct
0≤ 32 show that applied sample amount is suitable, and detected result is effective; Quality Control value Ct
0> 32 shows that applied sample amount is on the low side, again detects after can suitably improving applied sample amount;
With abrupt climatic change value Ct
1with Quality Control value Ct
0difference △ Ct whether there is the judging criterion of V600E sudden change as BRAF gene, △ Ct value>=9 detected result be negative, namely sample suddenly change without BRAF gene V600E or abundance of suddenling change lower than 0.1%, as shown in Figure 1; △ Ct value < 9 detected results are positive, and namely sample exists BRAF gene V600E sudden change, as shown in Figure 2.
In the present embodiment, the detected result of 50 routine clinical samples is as follows:
BRAF gene wild-type sample 6 example, 6 routine sample abrupt climatic change all without Ct value, as shown in Figure 3;
BRAF gene V600E sudden change sample 44 example, abrupt climatic change value Ct
1with Quality Control value Ct
0difference △ Ct < 10, one of them detected result is as shown in Figure 4.
Have 2 routine samples detection method of the present invention to be detected as BRAF gene V600E sudden change in above-mentioned detected result positive, and sequencing result is wild-type, after again repeat sequencing result and be indicated as this sample generation BRAF gene V600E and suddenly change, abundance of suddenling change is lower.All the other 42 routine sample sequencing results are consistent with fluorescence quantitative PCR detection result.
Above result shows that detection kit provided by the invention is for mankind BRAF gene V600E abrupt climatic change reliable results, be 95.5% with the coincidence rate of direct Sequencing, and detection method is highly sensitive in traditional sequencing methods, fast simple to operate, be beneficial to large-scale promotion.
The foregoing is only better embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and scope of the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (14)
1., for detecting the detection kit that mankind BRAF gene V600E suddenlys change, this test kit comprises the first pack and the second pack, described first pack and each self-contained PCR damping fluid of the second pack, dNTP, MgCl
2specific probe, lock nucleic acid primer, interior mark system, HotStartTaq enzyme, UNG enzyme, wherein the first pack comprises primer pair SEQIDNO:1 and SEQIDNO:2, probe SEQIDNO:3, second pack comprises primer pair SEQIDNO:1 and SEQIDNO:4, probe SEQIDNO:5, and wherein SEQIDNO:4 is lock nucleic acid primer.
2. the detection kit of mankind BRAF gene V600E sudden change as claimed in claim 1, it is characterized in that, 5 ' the end of probe SEQIDNO:3, SEQIDNO:5 is connected with fluorophor, and 3 ' end is connected with quenching group, and described fluorophor can be FAM, VIC or HEX.
3. the detection kit of mankind BRAF gene V600E sudden change as claimed in claim 1, it is characterized in that, described interior mark system comprises interior label primer and interior mark probe, and described interior label primer sequence is SEQIDNO:6 and SEQIDNO:7, and described interior mark probe sequence is SEQIDNO:8.
4. the mankind BRAF gene V600E as claimed in claim 3 detection kit of suddenling change, it is characterized in that, described interior mark probe 5 ' is terminal modified VIC, and 3 ' terminal modifiedly has TAMRA.
5. the mankind BRAF gene V600E as claimed in claim 1 detection kit of suddenling change, is characterized in that, all containing ROX reference corrected in the first pack and the second pack.
6. the detection kit of mankind BRAF gene V600E sudden change as claimed in claim 1, it is characterized in that, described detection kit contains positive control solution and blank liquid, and described positive control solution contains V600E mutant plasmid DNA, and described blank is Tris-HCl damping fluid.
7. a method for simple and quick detection mankind BRAF gene V600E sudden change, the method comprises the following steps:
(1) design and screen primer, the probe containing the Quality Control of mankind's BRAF gene, the detection probes containing BRAF gene V600E sudden change, ARMS primer, universal primer;
(2) testing sample genomic dna is obtained;
(3) detection kit according to any one of claim 1-6 is got, described genomic dna is successively got identical amount to add in described first reagent and described second reagent and to carry out PCR reaction simultaneously, the difference △ Ct of the Ct value obtained respectively according to two PCR reaction systems judges whether to have corresponding transgenation.
8. detect the method that mankind BRAF gene V600E suddenlys change, the method utilizes the detection kit described in any one of claim 1 to 6 to carry out, and the method comprises the following steps:
(1) testing sample genomic dna is obtained;
(2) genomic dna of above-mentioned acquisition successively being got same amount adds in the PCR reaction system containing the first pack in detection kit of the present invention and the PCR reaction system containing the second pack in detection kit of the present invention respectively, and make these two reaction systems carry out PCR reaction simultaneously, two PCR reaction is carried out independently of one another, and the difference △ Ct of the Ct value obtained respectively according to two PCR reaction systems judges whether to have corresponding transgenation.
9. method as claimed in claim 7 or 8, is characterized in that, carry out described judgement according to following condition:
There is BRAF gene V600E sudden change in △ Ct<9 then testing sample; △ Ct value >=9 testing sample without BRAF gene V600E sudden change or sudden change abundance lower than this test kit abrupt climatic change lower limit.
10. method as claimed in claim 7 or 8, it is characterized in that, also comprise the whether suitable step judged of applied sample amount, basis for estimation is as follows:
Ct
0≤ 32 applied sample amounts are suitable, and detected result is effective; Ct
0>32 then applied sample amount is on the low side, again detects after need increasing applied sample amount,
Wherein, described Ct
0for the Ct value that described first pack is corresponding.
11. methods as claimed in claim 8, is characterized in that, the situation of described △ Ct value >=9 comprises described second pack and carries out the rear situation without Ct value of PCR reaction.
12. methods according to any one of claim 7-11, is characterized in that, in described step (3), PCR reaction conditions is:
95℃5～10min；
95 DEG C of 15 ~ 30s, 60 DEG C of 45 ~ 60s, 40 ~ 45 circulations.
13. detection methods according to any one of claim 7-11, is characterized in that, in described step (3), PCR reaction conditions is preferably:
37℃10min；
95℃5min；
95 DEG C of 15s, 60 DEG C of 60s, 45 circulations.
14. methods as claimed in claim 8, is characterized in that, V600E detection system can also well be distinguished other mutagenesis template of V600, comprises V600K, V600D, V600R, V600M mutagenesis template.
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