CN105713987A - Primers, probe and kit for detecting human MET gene 14 exon splicing mutation - Google Patents
Primers, probe and kit for detecting human MET gene 14 exon splicing mutation Download PDFInfo
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Abstract
The invention discloses primers, a probe and a kit for detecting human MET gene 14 exon splicing mutation. The invention provides a specific primer pair A. The specific primer pair A is composed of a primer 1 and a primer 2 which are respectively disclosed as Sequence 3 and Sequence 4 in the sequence table. The invention also provides a primer-probe combination A which is composed of the specific primer pair A and a probe A, wherein the nucleotide sequence of the probe A is disclosed as Sequence 5 in the sequence table. The invention also provides a kit B which comprises the primer-probe combination A. The invention also provides application of the primer-probe combination A or the kit B in detecting whether mutation for causing MET gene 14 exon jump deletion occurs in the human genome DNA (deoxyribonucleic acid). The method is simple to operate, has the advantages of short detection time, high sensitivity, high specificity, high safety, no toxicity, low cost and the like, and can satisfy the actual demands for clinical quick detection.
Description
Technical field
The invention belongs to biotechnology and clinical molecular diagnosis technical field, relate to a kind of detecting the primer of mankind's MET gene 14 explicit leaming sudden change, probe and test kit.
Background technology
Pulmonary carcinoma is modal malignant tumor in world wide, and M & M occupies first of each cancer, and in cases of lung cancer, 80%-85% is nonsmall-cell lung cancer (nonsmallcelllungcancer, NSCLC).The annual whole world has more than the patient of 1,300,000 and dies from pulmonary carcinoma, and nearly half occurs in developing country.The sickness rate of China in Recent Years pulmonary carcinoma and case fatality rate are all in the notable trend raised, it is only 13% at China's patients with lung cancer survival rate of 5 years, its main cause is that most of pulmonary carcinoma is owing to lacking highly sensitive detection in Gene Mutation and making a definite diagnosis technology, and the therapeutic scheme of matched science, so that patient has missed the best opportunity for the treatment of.
At present, operation, radiation and chemotherapy are always up the primary treatments of NSCLC, but most chemotherapy can produce bigger toxic and side effects, and the chemotherapy effect between different patients has larger difference.In recent years, along with deepening continuously that Tumorigenesis and biological behaviour thereof are studied, the molecular targeted therapy of pulmonary carcinoma is high because of its specificity, and toxic and side effects is low, and being increasingly becoming clinical treatment field becomes study hotspot.C-met is a kind of protein product encoded by c-met proto-oncogene, for hepatocyte growth factor (hepatocytegrowthfactor, HGF) tyrosine kinase receptor, there is tyrosine kinase activity, membrane-proximal region part in its born of the same parents is encoded by MET gene 14 exon, comprises important adjustment element.The major way that HGF-MET abnormal signal activates includes: HGF process LAN, MET amplification, MET protein overexpression and MET gene mutation.It is now recognized that c-met is relevant to multiple oncoprotein and adjustment albumen, participates in the regulation and control of cellular informatics conduction, cytoskeleton rearrangement, be the key factor of cell proliferation, differentiation and motion.In July, 2014, american cancer gene studies group (TheCancerGenomeAtlas, that TCGA) has issued on Nature magazine that 230 examples can excise adenocarcinoma of lung comprises DNA, mRNA, the comprehensive gene analysis of spectrum result of miRNA level, by to mRNA and DNA high-flux sequence result and sequence alignment analysis, it has been found that the sudden change in MET gene DNA level the 14th exon montage region of 4% (10/230) adenocarcinoma of lung case causes that METEX14 occurs partially or completely jumping disappearance (exon14-skipping) in mRNA level in-site.Short 1 year half time after proposing is found at this, scientific research personnel with regard to METexon14-skipping deliver research more than 10, studies have found that MET gene 14 exon skipping disappearance is multiple and be born in nonsmall-cell lung cancer, and it is more common with lung carcinoma sarcomatodes therein and adenocarcinoma, incidence rate in adenocarcinoma of lung is about 3-4%, and the incidence rate in lung carcinoma sarcomatodes may be up to 22%.
MET14 explicit leaming suddenlys change, and suddenlys change with EGFR, and do not coexist phenomenon and the distinct Clinical symptoms containing this gene patient of K-ras sudden change, pointing out this target spot is the molecular marked compound that adenocarcinoma of lung specificity is higher.Small molecule tyrosine kinase inhibitors gram azoles is evident in efficacy to the patients with lung cancer containing MET gene mutation for Buddhist nun (Crizotinib), causes great interest and the research enthusiasm of people.In nonsmall-cell lung cancer NCCN (2016V2) guides in 2016, it is proposed that MET gene 14 explicit leaming sudden change patient-selectable gram azoles is for Buddhist nun.So far, MET inhibitor gram azoles has for the indication of Buddhist nun: ALK resets, MET expands, ROS-1 resets, the sudden change of MET14 explicit leaming.
At present, MET14 explicit leaming abrupt climatic change does not still have commercial kit, a few studies mechanism uses the method for high-flux sequence to carry out full exon scanning, it appeared that whether there is the sudden change of MET14 explicit leaming, the method is highly sensitive, tens even hundreds of tumor-related genes can be detected simultaneously, but complicated operation, later data processes loaded down with trivial details, the cost intensive of detection cycle length and detection, requirement for operation and interpretation technology is significantly high, clinical practice application still suffers from many deficiencies, present stage high-flux sequence there is no method and is applicable to extensive examination and the diagnosis of the MET positive NSCLC patient of sudden change for these reasons.
Summary of the invention
It is an object of the invention to provide and a kind of detect the primer of mankind's MET gene 14 explicit leaming sudden change, probe and test kit.
The present invention provides a species-specific primers to first, is made up of primer 1 and primer 2；
Described primer 1 is following (a1) or (a2):
(a1) single strand dna shown in sequence 3 of sequence table；
(a2) sequence 3 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 3；
Described primer 2 is following (a3) or (a4):
(a3) single strand dna shown in sequence 4 of sequence table；
(a4) sequence 4 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 4.
The purposes of first is following (c1) or (c2) by described special primer:
(c1) test kit is prepared；The function of described test kit is whether there occurs, in detection human gene group DNA, the sudden change causing MET gene 14 exon skipping to lack；
(c2) whether detection human gene group DNA there occurs the sudden change causing MET gene 14 exon skipping to lack.
The present invention also protects a kind of primed probe combination first, described special primer, first and probe first is made up of；
The nucleotides sequence of described probe first is classified as (b1) or (b2) as follows:
(b1) sequence 5 of sequence table；
(b2) replacement of one or several nucleotide and/or disappearance and/or interpolation have been carried out compared with sequence 5.
One end of described probe first has fluorescent reporter group, and another end has fluorescent quenching group.5 ' ends of described probe first have fluorescent reporter group, and 3 ' ends have fluorescent quenching group.Described fluorescent reporter group concretely FAM, described fluorescent quenching group concretely BHQ1.
The purposes of described primed probe combination first is following (c1) or (c2):
(c1) test kit is prepared；The function of described test kit is whether there occurs, in detection human gene group DNA, the sudden change causing MET gene 14 exon skipping to lack；
(c2) whether detection human gene group DNA there occurs the sudden change causing MET gene 14 exon skipping to lack.
The present invention also protects described special primer that first or described primed probe are combined the application of first, for as follows (c1) or (c2):
(c1) test kit is prepared；The function of described test kit is whether there occurs, in detection human gene group DNA, the sudden change causing MET gene 14 exon skipping to lack；
(c2) whether detection human gene group DNA there occurs the sudden change causing MET gene 14 exon skipping to lack.The present invention also protects a kind of test kit first, including described special primer to first.
The present invention also protects a kind of test kit first, including described special primer to first；The function of described test kit is whether there occurs, in detection human gene group DNA, the sudden change causing MET gene 14 exon skipping to lack.
The present invention also protects a kind of test kit second, combines first including described primed probe；The function of described test kit is whether there occurs, in detection human gene group DNA, the sudden change causing MET gene 14 exon skipping to lack.
Described test kit second also includes positive quality control plasmid；Described positive quality control plasmid is following (d1) or (d2):
(d1) sequence 9 containing ordered list is from the recombiant plasmid of the DNA molecular shown in 5 ' end 21-132 position nucleotide；
(d2) recombiant plasmid of the DNA molecular shown in sequence 9 containing ordered list.
In described (d1) or (d2), the plasmid concretely PCU57 carrier that sets out of described recombiant plasmid.
Described test kit second also includes primed probe combination second；The target behaviour ACTB gene of described primed probe combination second.
Second and probe second are made up of by described primed probe combination second special primer；
Second is made up of by described special primer primer 3 and primer 4；
Described primer 3 is following (e1) or (e2):
(e1) single strand dna shown in sequence 6 of sequence table；
(e2) sequence 6 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 6；
Described primer 4 is following (e3) or (e4):
(e3) single strand dna shown in sequence 7 of sequence table；
(e4) sequence 7 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 7；
The nucleotides sequence of described probe second is classified as (e5) or (e6) as follows:
(e5) sequence 8 of sequence table；
(e6) replacement of one or several nucleotide and/or disappearance and/or interpolation have been carried out compared with sequence 8.
One end of described probe second has fluorescent reporter group, and another end has fluorescent quenching group.5 ' ends of described probe second have fluorescent reporter group, and 3 ' ends have fluorescent quenching group.Described fluorescent reporter group concretely HEX, described fluorescent quenching group concretely BHQ1.
Described test kit second also includes internal control plasmid；Described internal control plasmid is following (f1) or (f2):
(f1) sequence 10 containing ordered list is from the recombiant plasmid of the DNA molecular shown in 5 ' end 136-212 position nucleotide；
(f2) recombiant plasmid of the DNA molecular shown in sequence 10 containing ordered list.
In described (f1) or (f2), the plasmid concretely PCU57 carrier that sets out of described recombiant plasmid.
Test kit described in any of the above may also include PCR premixed liquid and/or 2 × PCR buffer and/or the mixed liquid of enzyme and/or DEPC water.
2 × PCR buffer: solvent is the Tris-HCl buffer of pH8.0,50mM；Solute and concentration thereof is: dNTP2.5mM (refers to dATP, dTTP, dCTP, dGTP and is 2.5mM), MgCl250mM。
2 × PCR buffer of 12.5 μ L, primer 1, primer 2, primer 3, primer 4, probe first and probe second are mixed, obtains 20 μ LPCR premixed liquids.In 20 μ LPCR premixed liquids, the content of primer 1 is 0.8 μm of ol, and the content of primer 2 is 0.8 μm of ol, and the content of primer 3 is 0.8 μm of ol, and the content of primer 4 is 0.8 μm of ol, and the content of probe first is 0.4 μm of ol, and the content of probe second is 0.4 μm of ol.
Containing AMV reverse transcription and AMV-OptimizedTaq polymerase in the mixed liquid of enzyme.
Enzyme mixes liquid concretely Takara company, and article No. is the product of RR024A.
The present invention also protects described test kit first or described test kit second in the application that whether there occurs the sudden change causing MET gene 14 exon skipping to lack in detection human gene group DNA.
The present invention also protects a kind of method that whether there occurs the sudden change causing MET gene 14 exon skipping to lack in human gene group DNA of detection, comprises the steps:
(1) with the total serum IgE of patient to be measured for template, described primer 1, described primer 2, described primer 3, described primer 4, described probe first and described probe second is adopted to carry out real-time fluorescence quantitative PCR；Containing reverse transcription in the reaction system of described real-time fluorescence quantitative PCR；
(2) pcr amplification result is judged, it is judged that method is: if Ct value >=29 of the corresponding fluorescence signal of fluorescent reporter group second, represent the failure of an experiment；If the Ct value < 29 of the corresponding fluorescence signal of fluorescent reporter group second, judge according to following standard: if the Ct value < 39 of the corresponding fluorescence signal of fluorescent reporter group first, result is positive, and patient gene to be measured organizes in DNA and occurs or the doubtful sudden change causing MET gene 14 exon skipping to lack；If Ct value >=39 of the corresponding fluorescence signal of fluorescent reporter group first or without Ct value, result is negative, and patient gene to be measured organizes the sudden change not having to cause MET gene 14 exon skipping to lack in DNA.
The present invention also protects and a kind of detects the method whether sample to be tested exists the sudden change causing MET gene 14 exon skipping to lack, and comprises the steps:
(1) extracting the RNA of sample to be tested, reverse transcription is cDNA.
(2) cDNA obtained with step (1) is for template, adopts described primer 1, described primer 2, described primer 3, described primer 4, described probe first and described probe second to carry out real-time fluorescence quantitative PCR.
(3) pcr amplification result is judged, it is judged that method is: if Ct value >=29 of the corresponding fluorescence signal of fluorescent reporter group second, represent the failure of an experiment；If the Ct value < 29 of the corresponding fluorescence signal of fluorescent reporter group second, judge according to following standard: if the Ct value < 39 of the corresponding fluorescence signal of fluorescent reporter group first, result is positive, and patient gene to be measured organizes in DNA and occurs or the doubtful sudden change causing MET gene 14 exon skipping to lack；If Ct value >=39 of the corresponding fluorescence signal of fluorescent reporter group first or without Ct value, result is negative, and patient gene to be measured organizes the sudden change not having to cause MET gene 14 exon skipping to lack in DNA.
Human gene group DNA there occurs after causing the sudden change that MET gene 14 exon skipping lacks, transcribe the RNA obtained and there is the sequence 2 of sequence table from the nucleotide shown in 5 ' end 3018-3129 positions.The RNA that in human gene group DNA, MET genetic transcription obtains has the sequence 1 of sequence table from the nucleotide shown in 5 ' end 3018-3270 positions.Human gene group DNA there occurs after causing the sudden change that MET gene 14 exon skipping lacks, transcribe shown in the sequence 2 of the RNA such as sequence table obtained.Shown in the sequence 1 of the RNA that in human gene group DNA, MET genetic transcription obtains such as sequence table.
Sample to be tested described in any of the above is fresh pathological tissue, paraffin-embedded tissue or pleural fluid concretely.
The reaction system of quantitative fluorescent PCR described in any of the above is concretely: the total serum IgE mixing 20 μ LPCR premixed liquids, the mixed liquid of 1 μ L enzyme and 4 μ L steps 1 obtained.
The response procedures of quantitative fluorescent PCR described in any of the above is concretely: 50 DEG C of reaction 30min, 1 circulation；94 DEG C of reaction 2min, 1 circulation；94 DEG C of reaction 30s, 58 DEG C of reactions 30s, totally 44 circulations.
The invention provides a kind of MET14 explicit leaming mutation detection kit quickly cheap, easy and simple to handle and detection method.The method that the present invention sets up is simple to operate, and the detection time is short, it is only necessary to be directly added into the sample rna extracted, reverse transcription and pcr amplification carry out in the same reaction system closed, reducing pollution probability, reduce result simultaneously and the probability of deviation occur, one-time detection only needs complete for 90 minutes.This method has been provided simultaneously with highly sensitive, and specificity is good, safety non-toxic, and cost is low waits remarkable advantage, it is possible to meet the actual demand of clinical quickly detection.
Accompanying drawing explanation
Fig. 1 is positive quality control plasmid and internal control plasmid amplification curve chart
Fig. 2 is sensitivity technique result figure
Fig. 3 is specific detection result figure
Fig. 4 is the amplification curve diagram of clinical sample S13 (positive findings)
Fig. 5 is the amplification curve diagram of clinical sample S1 (negative findings)
Detailed description of the invention
Below example is easy to be more fully understood that the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and is commercially available from routine biochemistry reagent shop.Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged.
PCU57 carrier: Jin Sirui biotechnology, article No. SD1176.
Shown in the sequence 1 of the RNA such as sequence table that people's MET genetic transcription obtains.Transcribe after people's MET gene 14 exon skipping disappearance shown in the sequence 2 of the RNA such as sequence table obtained.Namely, after 14 exon skipping disappearances occur people MET gene, it is transcribed the RNA obtained and has lacked sequence 1 from 5 ' end 3078-3218 position nucleotide.
Embodiment 1, detection method are set up
One, design primer and probe
1, through a large amount of preliminary experiments and compliance test result, special primer is obtained to first and specific probe first.
First is made up of by special primer primer 1 and primer 2.
Primer 1 (sequence 3 of sequence table): 5 '-CACTGTTATTACTACTTGGGT-3 '；
Primer 2 (sequence 4 of sequence table): 5 '-AGAGGATACTGCACTTGTCG-3 '；
Following (sequence 5 of sequence table): the 5 '-AAGAGAAAGCAAATTAAAGATCAGT-3 ' of the nucleotide sequence of probe first；
5 ' ends of probe first have fluorescent reporter group FAM, and 3 ' ends have fluorescent quenching group BHQ1.
2, select people's ACTB gene as internal control gene, through preliminary experiment and compliance test result, obtain special primer to second and probe second.
Second is made up of by special primer primer 3 and primer 4.
Primer 3 (sequence 6 of sequence table): 5 '-GTCCACCTTCCAGCAGATG-3 '；
Primer 4 (sequence 7 of sequence table): 5 '-CCTAGAAGCATTTGCGG-3 '；
Following (sequence 8 of sequence table): the 5 '-GCAAGCAGGAGTATGACGAGTCCGG-3 ' of the nucleotide sequence of probe second；
5 ' ends of probe second have fluorescent reporter group HEX, and 3 ' ends have fluorescent quenching group BHQ1.
Two, test kit is prepared
Test kit is composed of the following components:
(1) PCR premixed liquid
2 × PCR buffer: solvent is the Tris-HCl buffer of pH8.0,50mM；Solute and concentration thereof is: dNTP2.5mM (refers to dATP, dTTP, dCTP, dGTP and is 2.5mM), MgCl250mM。
2 × PCR buffer of 12.5 μ L, primer 1, primer 2, primer 3, primer 4, probe first and probe second are mixed, obtains 20 μ LPCR premixed liquids.In 20 μ LPCR premixed liquids, the content of primer 1 is 0.8 μm of ol, and the content of primer 2 is 0.8 μm of ol, and the content of primer 3 is 0.8 μm of ol, and the content of primer 4 is 0.8 μm of ol, and the content of probe first is 0.4 μm of ol, and the content of probe second is 0.4 μm of ol.
(2) the mixed liquid of enzyme
Enzyme mixes liquid: Takara company, and article No. is RR024A.
Containing AMV reverse transcription and AMV-OptimizedTaq polymerase in the mixed liquid of enzyme.
(3) DEPC water
Three, the foundation of real-time fluorescence quantitative PCR detection method
Adopt test kit prepared by step 2 that testing sample is carried out real-time fluorescence quantitative PCR detection, specifically comprise the following steps that
1, the total serum IgE of sample to be tested is extracted.
2, real-time fluorescence quantitative PCR is carried out.
The reaction system (25 μ L) of real-time fluorescence quantitative PCR: the total serum IgE mixing that 20 μ LPCR premixed liquids, the mixed liquid of 1 μ L enzyme and 4 μ L steps 1 are obtained.
The response procedures of real-time fluorescence quantitative PCR: 50 DEG C of reaction 30min, 1 circulation；94 DEG C of reaction 2min, 1 circulation；94 DEG C of reaction 30s, 58 DEG C of reactions 30s, totally 44 circulations；FAM fluorescence signal (493/522nm) and HEX fluorescence signal (535-553nm) is detected when 58 DEG C.
If Ct value >=29 of HEX fluorescence signal, represent the failure of an experiment；
If the Ct value < 29 of HEX fluorescence signal, judge according to following standard: if Ct value >=39 of FAM fluorescence signal or without Ct value, result is negative, it was shown that do not have the sudden change causing MET gene 14 exon skipping to lack in the genomic DNA of this testing sample；If the Ct value < 39 of FAM fluorescence signal, result is positive, it was shown that there occurs the sudden change causing MET gene 14 exon skipping to lack in this testing sample genomic DNA.
Embodiment 2, detection method are verified
One, positive quality control plasmid and internal control plasmid are built
Positive quality control plasmid: the double chain DNA molecule shown in the sequence 9 of sequence table is inserted between BamI and the KpnI restriction enzyme site of PCU57 carrier, obtain recombiant plasmid (called after positive quality control plasmid).
Internal control plasmid: the double chain DNA molecule (partial sector in people's ACTB gene) shown in the sequence 10 of sequence table is inserted between XbaI and the KpnI restriction enzyme site of PCU57 carrier, the recombiant plasmid (called after internal control plasmid) obtained.
Two, the test kit that Application Example 1 obtains detects
1, positive quality control plasmid and internal control plasmid are mixed according to the ratio of copy number 10: 1, then adjust DNA concentration with Tris-HCl buffer (2.5mM, pH8.5), obtain the template solution that DNA concentration is 20ng/ μ L.
2, the template solution that step 1 obtains is adopted to carry out real-time fluorescence quantitative PCR.
The reaction system (25 μ L) of real-time fluorescence quantitative PCR: by 20 μ LPCR premixed liquids, the mixed liquid of 1 μ L enzyme and the mixing of 4 μ L template solution.
The response procedures of real-time fluorescence quantitative PCR: 50 DEG C of reaction 30min, 1 circulation；94 DEG C of reaction 2min, 1 circulation；94 DEG C of reaction 30s, 58 DEG C of reactions 30s, totally 44 circulations；FAM fluorescence signal (493/522nm) and HEX fluorescence signal (535-553nm) is detected when 58 DEG C.
Result is shown in Fig. 1.In Fig. 1, abscissa is period, and vertical coordinate is fluorescence signal intensity.It is shown that use the test kit of embodiment 1 preparation can realize the normal amplification of positive quality control plasmid and internal control plasmid.
Carrying out five repeated trials, result is consistent.
Embodiment 3, sensitivity, specificity and repeatability checking
The test kit of Example 1 preparation, carries out sensitivity, specificity and repeatability checking.
One, sensitivity
1, with the positive quality control plasmid of 10 times of gradient dilution embodiment 2 preparations of Tris-HCl buffer (2.5mM, pH8.5), each diluent is obtained.
2, take each diluent that step 1 obtains respectively as template, carry out real-time fluorescence quantitative PCR reaction.
The reaction system (25 μ L) of real-time fluorescence quantitative PCR: by 20 μ LPCR premixed liquids, the mixed liquid of 1 μ L enzyme and the mixing of 4 μ L diluents.
Diluent owing to adopting is different, forms reaction systems different as follows:
In reaction system 1, the initial content of positive quality control plasmid DNA is: 10000 copies；
In reaction system 2, the initial content of positive quality control plasmid DNA is: 1000 copies；
In reaction system 3, the initial content of positive quality control plasmid DNA is: 100 copies；
In reaction system 4, the initial content of positive quality control plasmid DNA is: 10 copies.
The response procedures of real-time fluorescence quantitative PCR: 50 DEG C of reaction 30min, 1 circulation；94 DEG C of reaction 2min, 1 circulation；94 DEG C of reaction 30s, 58 DEG C of reactions 30s, totally 44 circulations；FAM fluorescence signal (493/522nm) and HEX fluorescence signal (535-553nm) is detected when 58 DEG C.
Testing result is shown in Fig. 2.In Fig. 2, abscissa is period, and vertical coordinate is fluorescence signal intensity, and amplification curve from left to right is corresponding in turn to system 1-4.It is shown that the test kit of embodiment 1 preparation is highly sensitive, when the content of the positives Quality Control plasmid DNA of detection system is low to moderate 10-100 copy, it is also possible to obtain positive test symbol.
Carrying out five repeated trials, result is consistent.
Two, specificity
1, dilute the positive quality control plasmid of embodiment 2 preparation by normal person's Whole Blood Genomic DNA (concentration is 30ng/ μ l) as a setting, obtain each diluent.
2, take each diluent that step 1 arrives as template, carry out real-time fluorescence quantitative PCR reaction.
The reaction system (25 μ L) of real-time fluorescence quantitative PCR: by 20 μ LPCR premixed liquids, the mixed liquid of 1 μ L enzyme and the mixing of 4 μ L diluents.
Diluent owing to adopting is different, forms reaction systems different as follows:
In reaction system 1, the mutant proportion of positive quality control plasmid DNA is: 10%；
In reaction system 2, the mutant proportion of positive quality control plasmid DNA is: 5%；
In reaction system 3, the mutant proportion of positive quality control plasmid DNA is: 1%；
In reaction system 4, the mutant proportion of positive quality control plasmid DNA is: 0.5%；
In reaction system 5, the mutant proportion of positive quality control plasmid DNA is: 0.1%；
In reaction system 6, the mutant proportion of positive quality control plasmid DNA is: 0%；
The mutant proportion of positive quality control plasmid DNA is that in positive quality control plasmid, the copy number of target sequence accounts for the ratio of MET gene copy number in normal person's Whole Blood Genomic DNA.In 30ng normal person's Whole Blood Genomic DNA, the copy number of MET gene is about 10000.
The response procedures of real-time fluorescence quantitative PCR: 50 DEG C of reaction 30min, 1 circulation；94 DEG C of reaction 2min, 1 circulation；94 DEG C of reaction 30s, 58 DEG C of reactions 30s, totally 44 circulations；FAM fluorescence signal (493/522nm) and HEX fluorescence signal (535-553nm) is detected when 58 DEG C.
Testing result is shown in Fig. 3.In Fig. 3, abscissa is period, and vertical coordinate is fluorescence signal intensity, and amplification curve from left to right is corresponding in turn to system 1-6.It is shown that the detection method specificity that embodiment 1 is set up is good, when the content of the positives plasmid DNA of detection system is low to moderate 0.1%, it is also possible to substantially distinguish wild type sample.
Carrying out five repeated trials, result is consistent.
Three, repeatability
Experiment repeats 10 times, and Ct value differs less than 0.5 circulation, and detection method is reproducible.
Embodiment 4, clinical sample detect
Sample to be tested is: 45 example clinical diagnosises are the paraffin-embedded tissue sample (the median age is 61 years old for wherein male 29 example, women 16 example, is all obtain under the premise of patient's informed consent) of pulmonary carcinoma.It is S1-S45 by sample number consecutively.Each sample is proceeded as follows:
1, the total serum IgE of sample to be tested is extracted.
2, with the total serum IgE of step 1 for template, the detection method set up according to the step 3 of embodiment 1 carries out real-time fluorescence quantitative PCR.With the positive quality control plasmid of equal-volume embodiment 2 preparation as the positive control of total serum IgE.With the equal-volume DEPC water negative control as total serum IgE.
Testing result: in 45 example clinical samples, 44 examples are negative sample, and 1 example (S13) is positive sample, there occurs the sudden change causing MET gene 14 exon skipping to lack in its genomic DNA.The amplification curve of clinical sample S13 is shown in Fig. 4, Fig. 4, and abscissa is period, and vertical coordinate is fluorescence signal intensity.FAM and HEX signalling channel all has positive amplification curve.The amplification curve of clinical sample S1 (negative findings) is shown in Fig. 5, Fig. 5, and abscissa is period, and vertical coordinate is fluorescence signal intensity, and only HEX has positive amplification curve, the no positive amplification curve of FAM signalling channel.
3, total serum IgE reverse transcription step 1 obtained is cDNA, then adopts primer 1 and primer 2 to carry out pcr amplification, is checked order by pcr amplification product.The sequence 9 of the pcr amplification product of clinical sample S13 such as sequence table is from shown in 5 ' end 21-132 position nucleotide.Shown in the sequence 11 of the pcr amplification product of other 44 clinical samples such as sequence table.
It is shown that the coincidence rate of the testing result of the present invention and sequencing assay result is 100%, further demonstrate the accuracy of system of the present invention detection.
Claims (10)
1. special primer is to first, is made up of primer 1 and primer 2；
Described primer 1 is following (a1) or (a2):
(a1) single strand dna shown in sequence 3 of sequence table；
(a2) sequence 3 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 3；
Described primer 2 is following (a3) or (a4):
(a3) single strand dna shown in sequence 4 of sequence table；
(a4) sequence 4 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 4.
2. a primed probe combination first, described in claim 1, first and probe first are formed by special primer；
The nucleotides sequence of described probe first is classified as (b1) or (b2) as follows:
(b1) sequence 5 of sequence table；
(b2) replacement of one or several nucleotide and/or disappearance and/or interpolation have been carried out compared with sequence 5.
3. the special primer described in claim 1 is to first, or, the application of the primed probe combination first described in claim 2, for as follows (c1) or (c2):
(c1) test kit is prepared；The function of described test kit is whether there occurs, in detection human gene group DNA, the sudden change causing MET gene 14 exon skipping to lack；
(c2) whether detection human gene group DNA there occurs the sudden change causing MET gene 14 exon skipping to lack.
4. a test kit first, including special primer described in claim 1 to first；The function of described test kit is whether there occurs, in detection human gene group DNA, the sudden change causing MET gene 14 exon skipping to lack.
5. a test kit second, including primed probe combination first described in claim 2；The function of described test kit is whether there occurs, in detection human gene group DNA, the sudden change causing MET gene 14 exon skipping to lack.
6. test kit second as claimed in claim 5, it is characterised in that: described test kit second also includes positive quality control plasmid；Described positive quality control plasmid is following (d1) or (d2):
(d1) sequence 9 containing ordered list is from the recombiant plasmid of the DNA molecular shown in 5 ' end 21-132 position nucleotide；
(d2) recombiant plasmid of the DNA molecular shown in sequence 9 containing ordered list.
7. the test kit second as described in claim 5 or 6, it is characterised in that: described test kit second also includes primed probe combination second；The target behaviour ACTB gene of described primed probe combination second.
8. test kit first described in claim 4, or, the arbitrary described test kit second of claim 5-7, whether detection human gene group DNA there occurs the application of the sudden change causing MET gene 14 exon skipping to lack.
9. detect the method that whether there occurs the sudden change causing MET gene 14 exon skipping to lack in human gene group DNA, comprise the steps:
(1) with the total serum IgE of patient to be measured for template, primer 1, primer 2, primer 3, primer 4, probe first and probe second is adopted to carry out real-time fluorescence quantitative PCR；5 ' ends of described probe first have fluorescent reporter group first, and 3 ' ends have fluorescent quenching group；5 ' ends of described probe second have fluorescent reporter group second, and 3 ' ends have fluorescent quenching group；Containing reverse transcription in the reaction system of described real-time fluorescence quantitative PCR；
(2) pcr amplification result is judged, it is judged that method is: if Ct value >=29 of the corresponding fluorescence signal of fluorescent reporter group second, represent the failure of an experiment；If the Ct value < 29 of the corresponding fluorescence signal of fluorescent reporter group second, judge according to following standard: if the Ct value < 39 of the corresponding fluorescence signal of fluorescent reporter group first, result is positive, and patient gene to be measured organizes in DNA and occurs or the doubtful sudden change causing MET gene 14 exon skipping to lack；If Ct value >=39 of the corresponding fluorescence signal of fluorescent reporter group first or without Ct value, result is negative, and patient gene to be measured organizes the sudden change not having to cause MET gene 14 exon skipping to lack in DNA；
Described primer 1 is following (a1) or (a2):
(a1) single strand dna shown in sequence 3 of sequence table；
(a2) sequence 3 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 3；
Described primer 2 is following (a3) or (a4):
(a3) single strand dna shown in sequence 4 of sequence table；
(a4) sequence 4 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 4；
The nucleotides sequence of described probe first is classified as (b1) or (b2) as follows:
(b1) sequence 5 of sequence table；
(b2) replacement of one or several nucleotide and/or disappearance and/or interpolation have been carried out compared with sequence 5；
Described primer 3 is following (e1) or (e2):
(e1) single strand dna shown in sequence 6 of sequence table；
(e2) sequence 6 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 6；
Described primer 4 is following (e3) or (e4):
(e3) single strand dna shown in sequence 7 of sequence table；
(e4) sequence 7 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 7；
The nucleotides sequence of described probe second is classified as (e5) or (e6) as follows:
(e5) sequence 8 of sequence table；
(e6) replacement of one or several nucleotide and/or disappearance and/or interpolation have been carried out compared with sequence 8.
10. detect the method whether sample to be tested exists the sudden change causing MET gene 14 exon skipping to lack, comprise the steps:
(1) extracting the RNA of sample to be tested, reverse transcription is cDNA.
(2) cDNA obtained with step (1) is for template, adopts primer 1, primer 2, primer 3, primer 4, probe first and probe second to carry out real-time fluorescence quantitative PCR；5 ' ends of described probe first have fluorescent reporter group first, and 3 ' ends have fluorescent quenching group；5 ' ends of described probe second have fluorescent reporter group second, and 3 ' ends have fluorescent quenching group；
(3) pcr amplification result is judged, it is judged that method is: if Ct value >=29 of the corresponding fluorescence signal of fluorescent reporter group second, represent the failure of an experiment；If the Ct value < 29 of the corresponding fluorescence signal of fluorescent reporter group second, judge according to following standard: if the Ct value < 39 of the corresponding fluorescence signal of fluorescent reporter group first, result is positive, and patient gene to be measured organizes in DNA and occurs or the doubtful sudden change causing MET gene 14 exon skipping to lack；If Ct value >=39 of the corresponding fluorescence signal of fluorescent reporter group first or without Ct value, result is negative, and patient gene to be measured organizes the sudden change not having to cause MET gene 14 exon skipping to lack in DNA；
Described primer 1 is following (a1) or (a2):
(a1) single strand dna shown in sequence 3 of sequence table；
(a2) sequence 3 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 3；
Described primer 2 is following (a3) or (a4):
(a3) single strand dna shown in sequence 4 of sequence table；
(a4) sequence 4 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 4；
The nucleotides sequence of described probe first is classified as (b1) or (b2) as follows:
(b1) sequence 5 of sequence table；
(b2) replacement of one or several nucleotide and/or disappearance and/or interpolation have been carried out compared with sequence 5；
Described primer 3 is following (e1) or (e2):
(e1) single strand dna shown in sequence 6 of sequence table；
(e2) sequence 6 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 6；
Described primer 4 is following (e3) or (e4):
(e3) single strand dna shown in sequence 7 of sequence table；
(e4) sequence 7 through the replacement of one or several nucleotide and/or disappearance and/or interpolation and had the DNA molecular of identical function with sequence 7；
The nucleotides sequence of described probe second is classified as (e5) or (e6) as follows:
(e5) sequence 8 of sequence table；
(e6) replacement of one or several nucleotide and/or disappearance and/or interpolation have been carried out compared with sequence 8.
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