CN103305625A - Method and kit for detecting non-small cell lung cancer drive gene mutation spectrum, and application - Google Patents
Method and kit for detecting non-small cell lung cancer drive gene mutation spectrum, and application Download PDFInfo
- Publication number
- CN103305625A CN103305625A CN201310284338XA CN201310284338A CN103305625A CN 103305625 A CN103305625 A CN 103305625A CN 201310284338X A CN201310284338X A CN 201310284338XA CN 201310284338 A CN201310284338 A CN 201310284338A CN 103305625 A CN103305625 A CN 103305625A
- Authority
- CN
- China
- Prior art keywords
- mol
- egfr
- ex2f
- extf
- extension
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method and a kit for detecting a non-small cell lung cancer drive gene mutation spectrum, and an application. The method comprises the following steps of: designing 15 pairs of amplification primers for amplifying the exon segments of the seven related genes of non-small cell lung cancer, dividing the amplification primers into 6 groups, and preparing an amplification primer mixed solution, performing multiple PCR (polymerase chain reaction) amplification on the to-be-detected samples by the amplification primer mixed solution respectively, and then performing enzymatic digestion; and designing 39 extension primers used for detecting hotspot mutation sites, dividing the extension primers into 6 groups corresponding to the amplification primer mixed solution, and preparing an extension primer mixed solution, performing extension reaction on the digested PCR product, then performing enzymatic digestion, performing capillary electrophoresis on the obtained product, and making a result judgment via software analysis. The kit provided by the invention comprises the amplification primers for amplifying the exon segments of the seven related genes of non-small cell lung cancer, and the extension primers used for detecting hotspot mutation sites. The method and the kit provided by the invention are simple, high in flux, and short in time consumption.
Description
Technical field
The present invention relates to a kind of method that nonsmall-cell lung cancer drives gene mutation spectrum that detects, particularly a kind of method and test kit and application that detects nonsmall-cell lung cancer driving gene mutation spectrum.
Background technology
The sickness rate of lung cancer and case fatality rate all are positioned at the first place in tumour.Traditional methods for the treatment of is based on patient's Clinical symptoms and histological type is selected the chemotherapy scheme, but only has 6 months without the progression of disease phase (PFS).In recent years, the targeted therapy of lung cancer makes remarkable progress, the most outstanding example is: the small molecule tyrosine kinase inhibitors take Urogastron (EGFR) as target spot demonstrates significant curative effect in the Patients with Non-small-cell Lung that contains mutant egf R, and PFS has reached 9-13 month.Subsequently, the gram azoles take the EML4-ALK fusion gene as target spot also demonstrates same clinical efficacy for the Buddhist nun in the targeted therapy of nonsmall-cell lung cancer.Obviously, the single pathological of lung cancer can not meet clinical needs, needs to understand molecule and the molecule mechanism thereof that plays a crucial role generation in lung cancer, the evolution in depth from gene level.Studies show that, can according to the molecular mutation state of lung cancer, nonsmall-cell lung cancer be divided into different molecule subgroups.Progress over nearly 10 years has been found the target spot of many pharmacological agenies, and successively bibliographical information has been arranged nonsmall-cell lung cancer drive gene mutation spectrum, surpass 80% adenocarcinoma of lung, approximately 47% lung squamous cancer is with the sudden change that drives gene, for the targeted therapy of nonsmall-cell lung cancer provides molecular basis.
Except the small molecule tyrosine kinase inhibitors take EGFR, EML4-ALK fusion gene as target spot clinical application, PI3K inhibitor, BRAF suppress, mek inhibitor has all entered clinical experimental stage, therefore, be badly in need of a kind of test kit of exploitation or method and provide the disposable detection of carrying out the lung cancer gene mutation spectrum for clinical, so that for clinical trial screening patient or provide the guidance foundation for the patient selects methods for the treatment of in the future.
Unit of the present invention is in earlier stage to the gene studies data presentation such as EGFR, KRAS, PIK3CA, BRAF, PTEN, MEK1 in state's Non-small cell lung carcinoma, and gook and westerner's nonsmall-cell lung cancer molecular mutation spectrum there are differences, and is as shown in table 1.The mutation rate of EGFR in gook and westerner is respectively 47.9% and 19.2%; KRAS is respectively 26.1% and 11.2%.
Table 1 nonsmall-cell lung cancer drives the hot spot mutation of gene
| The gene title | The nucleic acid site | Amino acid | The gene title | The nucleic acid site | Amino acid |
| BRAF | 1397G>T | G466V | KRAS | 34G>T/A/C | G12C/S/R |
| 1406G>C | G469A | 35G>T/A/C | G12V/D/A | ||
| 1789C>G | L596V | 37G>T/A/C | G13C/S/R | ||
| 1798G>A | V600K | 38G>C/A/T | G13A/D/V | ||
| 1799T>G/A | V600G/E | PTEN | 388C> | R130 | |
| 517C>T | R173C | ||||
| PIK3CA | 1624G>A | E542K | 697C>T | R233X | |
| 1633G>C/A | E545Q/K | 800A>G | K267fs*9 | ||
| 1636C>A | Q546K | EGFR | 2155G>A/T | G719S/C | |
| 1637A>G | Q546R | 2156>A/C | G719D/A | ||
| 3139C>T | H1047Y | 2235_2249del15 | E746_A750(1) | ||
| 3140A>T/G | H1047L/R | 2236_2250del15 | E746_A750(2) | ||
| 3145G>A/C | G1049S/R | 2369C>T | T790M | ||
| NRAS | 34G>C/T/G | G12R/C/S | 2573T>C | L858R | |
| 35G>A/C/T | G12D/A/V | 2582T>A | L861Q | ||
| 37G>T/A/C | G13C/S/R | ||||
| 38G>C/A/T | G13A/D/V | MEK1 | 167A>C | Q56P | |
| 181C>A/G | Q61K/E | 171G>T | K58N |
| 182A>C/T/G | Q61P/L/R | 199G>A | D67N | ||
| 183A>T/C | Q61H |
At present, the common detection method of transgenation has: direct sequencing, ARMS method, HRM method, DHPLC method etc., the common shortcoming of these methods is to detect the sudden change of a sudden change or a fragment gene at every turn, the detection flux is low, examination drives the mutation status of gene one by one, and patient waits for that result's time is longer.Simultaneously, the difficulty because end-stage patients draw materials, sample size is less, often can not satisfy the needs that aforesaid method detects gene mutation spectrum.Though degree of depth sequence measurement can meet clinical needs at flux, because expensive can not in clinical application, popularizing.
Summary of the invention
The shortcoming that primary and foremost purpose of the present invention is to overcome prior art provides a kind of method that nonsmall-cell lung cancer drives gene mutation spectrum that detects with not enough.Use multiplex PCR and detect nonsmall-cell lung cancer driving gene mutation spectrum in conjunction with the single-basic extension method.The driving gene of the application's indication is seven kinds of EGFR, BRAF, PIK3CA, PTEN, MEK1, KRAS and NRAS etc. and non-small cell lung oncogenesis and keeps genes involved.
Another object of the present invention is to provide the test kit that detects nonsmall-cell lung cancer driving gene mutation spectrum.This test kit is for obtaining according to the aforesaid method design.
A further object of the present invention is to provide the application of mentioned reagent box.
Purpose of the present invention is achieved through the following technical solutions: a kind of method that detects nonsmall-cell lung cancer driving gene mutation spectrum comprises following steps:
(1) amplimer of seven kinds of genes involved exons of design amplification nonsmall-cell lung cancer section
The amplimer of designed seven kinds of genes involved exons of amplification nonsmall-cell lung cancer section is as shown in table 2:
The amplimer (5'-3') of each genes involved exon section of table 2
Increase with the exon DNA section that comprises hot mutant site among the primer pair nonsmall-cell lung cancer shown in the table 2 seven kinds of related gene B RAF, EGFR, PI3K, PTEN, MEK1, KRAS and NRAS, wherein, the exon section of BRAF is BRAF_EX11, BRAF_EX15; The exon section of EGFR is EGFR_EX18, EGFR_EX19, EGFR_EX20, EGFR_EX21; The exon section of KRAS is KRAS_EX2; The exon section of NRAS is NRAS_EX2, NRAS_EX3; The exon section of PI3K is PIK3K_EX9, PIK3K_EX20; The exon section of PTEN is PTEN_EX5, PTEN_EX6, PTEN_EX7; The exon section of MEK1 is MEK1_EX2;
(2) multiplex PCR amplification
Seven amplimers that drive the gene extron sub-segments of amplification nonsmall-cell lung cancer of step (1) design are mixed with the amplimer mixed solution, are divided into 6 groups such as P1, P2, P3, P4, P5, P6, as shown in table 3:
Seven preparations that drive the amplimer mixed solution of gene extron sub-segments of table 3
Use respectively amplimer mixed solution P1, P2, P3, P4, P5, P6 to carry out the multiplex PCR amplification, obtain the PCR product;
(3) PCR product digestion: the PCR product that step (2) obtains is through alkaline phosphatase and exonuclease digestion; The digestion system is: PCR product 2 μ l, exonuclease 0.5 μ l, alkaline phosphatase 1 μ l, H
2O6.5 μ l; Digestion condition is: 37 ℃ 60 minutes, 75 ℃ 15 minutes;
(4) be designed for the extension primer that detects hot mutant site: be designed for the extension primer of single-basic extension method for the hot spot mutation of the driving of the nonsmall-cell lung cancer described in the table 1 gene, 39 extension primer sequences for detection of hot mutant site of seven nonsmall-cell lung cancer driving genes are as shown in table 4:
Table 4 is for detection of the extension primer (5'3') of hot mutant site
(5) extension: will extend primer by table 5 and be mixed with the primer mixed solution, corresponding to above-mentioned PCR group, it extends primer sets and is respectively E1, E2, E3, E4, E5, E6, and the SNaPshot Multiplex Ready Reaction Mix enzyme that adopts AB company to produce carries out respectively extension to the postdigestive PCR product of step (3);
Table 5 extends the preparation of primer mixed solution
(6) enzymic digestion: in the extension product, add alkaline phosphatase and digest;
(7) capillary electrophoresis: postdigestive extension products is got 1 μ l, adds 9 μ l methane amides, 0.2 μ l LIZ120, mixing, carry out capillary electrophoresis at AB3730 genetic analysis instrument, electrophoresis result draws the sudden change situation of each goal gene with the GeneMapper4.1 software analysis, makes the result and judges;
(8) result judges: 1) according to the location positioning gene locus at electrophoresis peak; 2) determine the base type according to the color at electrophoresis peak: blueness represents the G base, green represents the A base, redness represents the T base, black represents the C base;
The condition of the described multiplex PCR of step (2) amplification is: 95 ℃ 5 minutes; (95 ℃ 20 seconds, 58 ℃ 30 seconds, 72 ℃ 1 minute), 40 circulations; 72 ℃ 3 minutes;
The system of the described multiplex PCR amplification of step (2) is: Premix Hotstart Taq5 μ l, amplimer mixed solution 1 μ l, dna profiling 20ng, and distilled water is mended to 10 μ l;
The system of the described extension of step (5) is: SNaPshot Multiplex Ready Reaction Mix2 μ l, postdigestive PCR product 2 μ l, extension primer mixed solution 1 μ l, H
2O5 μ l;
The condition of the described extension of step (5) is: 96 ℃ 30 seconds; (96 ℃ 10 seconds, 50 ℃ 10 seconds, 60 ℃ 30 seconds), 35 circulations;
The condition of the described enzymic digestion of step (6) is: extension product 9 μ l, alkaline phosphatase 1 μ l; 37 ℃ of digestion in 60 minutes, 75 ℃ of 15 minutes remaining enzymes of deactivation;
The operational condition of the described capillary electrophoresis of step (7) is AB3730 genetic analysis instrument user manual Standard operation procedure SOP;
A kind of test kit that detects nonsmall-cell lung cancer driving gene mutation spectrum, for the method design that drives gene mutation spectrum according to above-mentioned detection nonsmall-cell lung cancer obtains, comprise amplimer and the listed extension primer for detection of hot mutant site of table 4 of listed seven kinds of genes involved exons of the amplification nonsmall-cell lung cancer section of table 2;
Described detection nonsmall-cell lung cancer drives the test kit of gene mutation spectrum, preferably comprises by the amplimer mixed solution of the listed P1 of table 3, P2, P3, P4, P5, P6 and by E1, E2, E3, E4, E5, the E6 of table 5 preparation to extend the primer mixed solution;
Described detection nonsmall-cell lung cancer drives the test kit of gene mutation spectrum, preferably also comprises Premix Hotstart Taq, alkaline phosphatase and exonuclease and SNaPshot Multiplex Ready Reaction Mix enzyme;
Described detection nonsmall-cell lung cancer drives the test kit of gene mutation spectrum and uses in clinical as diagnostic reagent, drives gene mutation spectrum for detection of nonsmall-cell lung cancer;
Described detection nonsmall-cell lung cancer drives the using method of the test kit of gene mutation spectrum, may further comprise the steps:
1. carry out respectively pcr amplification with amplimer mixed solution P1, P2, P3, P4, P5, P6, obtain the PCR product; The pcr amplification condition is: 95 ℃ 5 minutes; (95 ℃ 20 seconds, 58 ℃ 30 seconds, 72 ℃ 1 minute), 40 circulations; 72 ℃ 3 minutes; The pcr amplification system is: Premix Hotstart Taq5 μ l, amplimer mixed solution 1 μ l, dna profiling 20ng, and distilled water is mended to 10 μ l;
2. the PCR product that 1. step is obtained is through alkaline phosphatase and exonuclease digestion; The digestion system is: PCR product 2 μ l, exonuclease 0.5 μ l, alkaline phosphatase 1 μ l, H
2O6.5 μ l; Digestion condition is: 37 ℃ 60 minutes, 75 ℃ 15 minutes;
3. extension: will extend primer by table 5 and be mixed with and extend the primer mixed solution, and be respectively E1, E2, E3, E4, E5, E6; The extension system is: SNaPshot Multiplex Ready Reaction Mix2 μ l, PCR product 2 μ l, extension primer mixed solution (being respectively E1, E2, E3, E4, E5, E6) 1 μ l, H
2O5 μ l; The extension condition is: 96 ℃ 30 seconds; (96 ℃ 10 seconds, 50 ℃ 10 seconds, 60 ℃ 30 seconds), 35 circulations;
4. the alkaline phosphatase that adds 1 μ l in the every pipe PCR product that 3. step is obtained, 37 ℃ of digestion in 60 minutes, 75 ℃ of 15 minutes remaining enzymes of deactivation;
5. postdigestive extension products is got 1 μ l, adds 9 μ l methane amides, 0.2 μ l LIZ120, and mixing carries out capillary electrophoresis at AB3730 genetic analysis instrument; Electrophoresis result draws the sudden change situation of each goal gene with the GeneMapper4.1 software analysis, make the result and judge.
The present invention has following advantage and effect with respect to prior art:
(1) flux is high: existing detection method of gene mutation step loaded down with trivial details (such as direct sequencing), once can only detect several sites (such as HRM, ARMS, DHPLC) of a gene, flux is lower; Method provided by the invention and test kit once can detect 7 of nonsmall-cell lung cancers and drive the gene sudden change in the individual site of totally 37 (wherein two of EGFR_EXON19 deletion mutantions detect from positive and negative both direction respectively); The present invention is with respect to direct Sequencing and ARMS detection technique, and number gene and mutational site that single detects are many.
(2) step is simple: method provided by the invention and test kit are with respect to direct Sequencing technology and DHPLC technology, and operation steps is simple, and the result easily judges.
(3) time is short: because method provided by the invention and test kit once can detect the hot spot mutation of a plurality of genes, finishing 7 detections that drive gene only needs two working dayss, 140ng DNA, and the direct Sequencing rule needs three working dayss, 300ng DNA.Both can shorten detection time, can greatly save again the usage quantity of DNA.
Description of drawings
Fig. 1 is the result that embodiment 1 sample uses extension primer mixed solution E1 to obtain: the capillary electrophoresis figure that is respectively from left to right KRAS_34, EGFR_2235, EGFR_2369, NRAS_37, NRAS_181 and six sites of PIK3CA_1633, the result is respectively C, G, G, G, C, G, is wild-type; X-coordinate represents the extension products size, and ordinate zou represents fluorescence intensity.
Fig. 2 is the as a result figure that embodiment 1 sample uses extension primer mixed solution E2 to obtain: be respectively from left to right the capillary electrophoresis figure in EGFR_2235R, NRAS_38, BRAF_1799, NRAS_182 and five sites of MEK1_199, the result is respectively G, C, T, A, C; X-coordinate represents the extension products size, and ordinate zou represents fluorescence intensity.
Fig. 3 is the as a result figure that embodiment 1 sample uses extension primer mixed solution E3 to obtain: be respectively from left to right the capillary electrophoresis figure in KRAS_35, EGFR_2236R, EGFR_2573, BRAF_1798, PIK3CA_1624, PIK3CA_1637 and seven sites of NRAS_35, the result is respectively G, G, T/G, G, C, A, G; T occurs in results suggest EGFR Nucleotide 2369 sites〉the G sudden change, the L858R sudden change occurs in amino acid; X-coordinate represents that extension products size, ordinate zou represent fluorescence intensity.
Fig. 4 is the as a result figure that embodiment 1 sample uses extension primer mixed solution E4 to obtain: be respectively from left to right the capillary electrophoresis figure in MEK1_171, PIK3CA_3140, EGFR_2236R, PTEN_517, EGFR_2156 and six sites of PIK3CA3139, the result is respectively C, T, T, C, G, G; X-coordinate represents the extension products size, and ordinate zou represents fluorescence intensity.
Fig. 5 is the as a result figure that embodiment 1 sample uses extension primer mixed solution E5 to obtain: the capillary electrophoresis figure that is respectively from left to right EGFR_2582, KRAS_37, MEK1_167, EGFR_2155, BRAF_1406, PIK3CA_3145, NRAS_183 and eight sites of BRAF_1789, the result is respectively A, G, A, G, G, C, T, C, is wild-type; X-coordinate represents the extension products size, and ordinate zou represents fluorescence intensity.
Fig. 6 is the as a result figure that embodiment 1 sample uses extension primer mixed solution E6 to obtain: be respectively from left to right the capillary electrophoresis figure in NRAS_34, KRAS_38, PTEN_388, PTEN_697, PTEN_800, PIK3CA_1636 and seven sites of BRAF_1397, the result is respectively C, G, C, G, A, C, G; X-coordinate represents the extension products size, and ordinate zou represents fluorescence intensity.
Fig. 7 is the as a result figure that Comparative Examples 1 sample uses the extension primer mixed solution E2 before the primer concentration optimization to obtain: be respectively from left to right the capillary electrophoresis figure in EGFR_2235R, NRAS_38, BRAF_1799, NRAS_182 and five sites of MEK1_199, the result be respectively G, C, T ,/, C; Wherein BRAF_1799 electrophoresis peak is on the low side, and NRAS_182 does not go out the peak; X-coordinate represents the extension products size, and ordinate zou represents fluorescence intensity.
Fig. 8 is the as a result figure that sample uses the front extension primer mixed solution E6 of combination of primers optimization to obtain in the Comparative Examples 2: be respectively from left to right the capillary electrophoresis figure in NRAS_34, KRAS_35, PTEN_388, PTEN_697, PIK3CA_1636 and six sites of BRAF_1397, the result is respectively C, G, C, G, C, G; Results suggest, KRAS Nucleotide 35 site bases are G, wild-type; X-coordinate represents the extension products size, and ordinate zou represents fluorescence intensity.
Fig. 9 is that Comparative Examples 2 samples use combination of primers optimization to extend afterwards the as a result figure that primer mixed solution E3 obtains: be respectively from left to right the capillary electrophoresis figure in KRAS_35, EGFR_2236R, EGFR_2573, BRAF_1798, PIK3CA_1624, PIK3CA_1637 and seven sites of NRAS_35, the result is respectively G/A, G, T, G, C, A, G; G occurs in results suggest KRAS Nucleotide 35 sites〉the A sudden change; X-coordinate represents that extension products size, ordinate zou represent fluorescence intensity.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
Embodiment 1
(1) design of primers
(1) seven amplimers that drive the gene extron sub-segments of design amplification nonsmall-cell lung cancer
Driving the exon DNA section that comprises hot mutant site among gene BRAF, EGFR, PIK3CA, PTEN, MEK1, KRAS and the NRAS with seven of primer pair nonsmall-cell lung cancers increases, wherein, the exon section of BRAF is BRAF_EX11, BRAF_EX15; The exon section of EGFR is EGFR_EX18, EGFR_EX19, EGFR_EX20, EGFR_EX21; The exon section of KRAS is KRAS_EX2; The exon section of NRAS is NRAS_EX2, NRAS_EX3; The exon section of PIK3CA is PIK3CA_EX9, PIK3CA_EX20; The exon section of PTEN is PTEN_EX5, PTEN_EX6, PTEN_EX7; The exon section of MEK1 is MEK1_EX2.
Seven amplimers that drive the gene extron sub-segments of designed amplification nonsmall-cell lung cancer are as shown in table 6:
The amplimer (5'-3') of each genes involved exon section of table 6
(2) design extension primer: be designed for the extension primer of single-basic extension method for the hot spot mutation of the driving of the nonsmall-cell lung cancer described in the table 1 gene, seven nonsmall-cell lung cancers drive 39 extension primer sequence such as tables 7 for detection of hot mutant site of genes.
Table 7 is for detection of the extension primer (5'-3') of hot mutant site
(3) preparation of PCR primer mixed solution: by table 8 the pcr amplification primer is mixed with mixed solution, is respectively P1, P2, P3, P4, P5, P6 totally 6 groups, as shown in table 8.
Seven preparations that drive the amplimer mixed solution of gene extron sub-segments of table 8
(4) preparation of extension primer mixed solution: will extend primer by table 9 and be mixed with mixed solution, be respectively 6 groups of E1, E2, E3, E4, E5, E6 etc., correspond respectively to above-mentioned P1, P2, P3, P4, P5, P6 combination, as shown in table 9.
Table 9 extends the preparation of primer mixed solution
(2) pattern detection
(1) multiplex PCR amplification
Using-system DNA extraction test kit (Hua Shun company) is pressed the test kit operation and is told a story bright to an operation of lung cancer resection organization sample (Guangdong, called after sample 1) behind the extraction DNA, carry out the multiplex PCR amplification with amplimer mixed solution P1, P2, P3, P4, P5, P6 respectively, the 1 pipe normal human blood genomic dna that increases simultaneously (extracting DNA by a day root company blood sample DNA extraction test kit specification sheets operation) is as normal control.The pcr amplification condition is: 95 ℃ 5 minutes; (95 ℃ 20 seconds, 58 ℃ 30 seconds, 72 ℃ 1 minute), 40 circulations; 72 ℃ 3 minutes.The PCR system of P1 pipe is: 5 μ lPremix Hotstart Taq(Takara), 1 μ l amplimer mixed solution P1,1 μ l(20ng/ μ l) DNA, 3 μ l H
2O.All the other P2, P3, P4, P5, all according to said method preparations of P6 pipe PCR reaction system.
(2) PCR product digestion: the PCR product that step (1) is obtained digests, and the digestion system is: PCR product 2 μ l, exonuclease (Takara) 0.5 μ l, alkaline phosphatase (Takara) 1 μ l, H
2O6.5 μ l; Digestion condition be 37 ℃ 60 minutes, 75 ℃ 15 minutes.Obtain respectively P1, P2, P3, P4, P5, P6 digestion product.
2 μ l SNaPshot Multiplex Ready Reaction Mix(American AB companies), the P1 digestion product of 2 μ l steps (2), 1 μ l E1 extend primer mixture, 5 μ l H (3) extension: E1 extension system:
2O.All the other E2, E3, E4, E5, E6 extension system are according to said method prepared (being the corresponding E6 of corresponding E5, P6 of corresponding E4, P5 of corresponding E3, P4 of P2 corresponding E2, P3); The extension condition is: 96 ℃ 30 seconds; 96 ℃ 10 seconds, 50 ℃ 10 seconds, 60 ℃ 30 seconds, 35 circulations.
(4) enzymic digestion: in every pipe extension product, add the alkaline phosphatase (Takara) of 1 μ l, 37 ℃ of digestion in 60 minutes, 75 ℃ of 15 minutes remaining enzymes of deactivation.
(5) postdigestive extension products is got 1 μ l, adds 9 μ l methane amides, 0.2 μ l LIZ120, and mixing carries out electrophoresis at AB3730 genetic analysis instrument.Adopt the GeneMaper4.1 analytical results.
(3) detected result: the detected result of sample 1 is shown in Fig. 1～6: Fig. 1,2,4,5 and 6 result represent that the site of detecting is wild-type, the result of Fig. 3 shows in the E3 combination, T occurs in EGFR gene nucleotide 2369 sites〉the G sudden change, the L858R sudden change occurs in amino acid, and all the other gene locuss are wild-type.Use direct sequencing that sample 1 is analyzed, the result who obtains is consistent with result of the present invention.The result that the normal human blood genome obtains represents that the site of detecting is wild-type.As seen method provided by the invention and test kit can detect accurately and efficiently nonsmall-cell lung cancer and drive transgenation.
Comparative Examples 1
Operation steps is basic with condition and embodiment 1 is consistent, difference only is: the concentration that the E2 group is extended primer changes, be specially BRAF1799_extF and become 2pmol/L by 3pmol/L, NRAS182_extF becomes 1pmol/L by 3pmol/L, and other three extension primer concentrations are constant.As a result, the electrophoresis peak of BRAF1799_extF obviously reduces, and NRAS182_extF electrophoresis peak disappears, as shown in Figure 7.This Comparative Examples is intended to explanation, in the situation that other conditions are constant, extends in the mixture of primer, and the concentration ratio of each primer composition is a key factor of experiment success or failure.
This Comparative Examples explanation, when carrying out multiplex PCR and extension, in the primer mixture, exist the effect of vying each other between the various primers, the reaction efficiency of some primer is high, and the peak shape of generation is higher, but some primer efficient then is suppressed, so the concentration of various primers and ratio were very large on result's impact during primer mixed, need constantly to adjust, obtain best primer concentration ratio.
Comparative Examples 2
(1) design of primers
(1) seven amplimers that drive the gene extron sub-segments of design amplification nonsmall-cell lung cancer: with embodiment 1.
(2) design extension primer: with embodiment 1.
(3) preparation of PCR primer mixed solution: as shown in table 10.
Seven preparations that drive the amplimer mixed solution of gene extron sub-segments of table 10
(4) preparation of extension primer mixed solution: KRAS35_extF extension primer is adjusted to the E6 group from the E3 group, and is as shown in table 11.
Table 11 extends the preparation of primer mixed solution
(2) pattern detection
Sample 2: from the DNA of a patients with lung cancer puncture tissue samples (Guangdong) extraction.
Carry out DNA extraction, pcr amplification, enzymic digestion, extension and interpretation of result by embodiment 1 condition and method.
The result:
This Comparative Examples purpose is to illustrate that extending primer KRAS35 is positioned at different groups, extend efficient different, therefore, detected result for sample 2 other genes is not done to show and explanation at this, only show and illustrate the result in KRAS35 site, see Fig. 8: second base peak figure that the peak is KRAS gene nucleotide 35 sites among the figure, be the G peak, namely be wild-type.
Fully according to condition and the method repeated experiments of embodiment 1, the results are shown in Figure 9: first peak is KRAS35 base peak figure among the figure with sample 2, and the result shows, KRAS gene nucleotide 35 sites generation G〉the A sudden change.The result is consistent with the result that direct survey method obtains.
As seen, exist the effect of vying each other between the primer, need to optimize the combination of each primer, can obtain the high result of accuracy.
Effect embodiment
The sample that detects 110 routine tumour patients by operation steps and the condition of embodiment 1, and with direct sequencing relatively, sensitivity of the present invention is 100%, specific degree is 98.4%, positive predictive value is 97.9%, negative predictive value is 100%.Referring to table 10.
Table 10 sensitivity and specific degree
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1. one kind is detected the method that nonsmall-cell lung cancer drives gene mutation spectrum, it is characterized in that comprising following steps:
(1) amplimer of seven kinds of genes involved exons of design amplification nonsmall-cell lung cancer section, as follows:
BRAF_EX11F：5'-TGTTTGGCTTGACTTGACT-3'；
BRAF_EX11R：5'-ACAATGTCACCACATTACAT-3'；
BRAF_EX15F：5'-GCTCTGATAGGAAAATGAGAT-3'；
BRAF_EX15R：5'-CTGATGGGACCCACTCCAT-3'；
EGFR_EX18F：5'-CTCCCAACCAAGCTCTCTTGA-3'；
EGFR_EX18R：5'-CCTTATACACCGTGCCGAAC-3'；
EGFR_EX19F：5'-GATCCCAGAAGGTGAGAAAG-3'；
EGFR_EX19R：5'-AAGCAGAAACTCACATCGAG-3'；
EGFR_EX20F：5'-ATCTGCCTCACCTCCACCGT-3'；
EGFR_EX20R：5'-TGGACTATGTCCGGGAACAC-3'；
EGFR_EX21F：5'-AACACCGCAGCATGTCAAGATC-3'；
EGFR_EX21R：5'-TGCCTCCTTCTGCATGGTATT-3'；
KRAS_EX2F：5'-CATTATTTTTATTATAAGGCCTG-3'；
KRAS_EX2R：5'-AGAATGGTCCTGCACCAGTA-3'；
NRAS_EX2F：5'-CAACAGGTTCTTGCTGGTGT-3'；
NRAS_EX2R：5'-GTAAAGATGATCCGACAAGTGAGAG-3'；
NRAS_EX3F：5'-TGGTGAAACCTGTTTGTTGG-3'；
NRAS_EX3R：5'-CCTTTCAGAGAAAATAATGCTC-3'；
PIK3CA_EX9F：5'-ATGACAAAGAACAGCTCAAAGCA-3'；
PIK3CA_EX9R：5'-TCCATTTTAGCACTTACCTGTG-3'；
PIK3CA_EX20F：5'-GAGCAAGAGGCTTTGGAGTA-3'；
PIK3CA_EX20R：5'-ATCCAATCCATTTTTGTTGTCC-3'；
PTEN_EX5F：5'-TATTCTGAGGTTATCTTTTTACCAC-3'；
PTEN_EX5R：5'-GTGCACATATCATTACACCAGTTC-3'；
PTEN_EX6F：5'-TTTTCTGTCCACCAGGGAGT-3'；
PTEN_EX6R：5'-TCCAGATGATTCTTTAACAGGTAG-3'；
PTEN_EX7F：5'-GAAGATATATTCCTCCAATTCA-3'；
PTEN_EX7R：5'-TTCTCCCAATGAAAGTAAAGTACA-3'；
MEK1_EX2F：5'-GCTTGATGAGCAGCAGCGAAA-3'；
MEK1_EX2R：5'-CACCACACCGCCATTGCCAGC-3'；
(2) multiplex PCR amplification
Seven amplimers that drive the gene extron sub-segments of amplification nonsmall-cell lung cancer of step (1) design are mixed with the amplimer mixed solution, are divided into 6 groups, as follows:
P1 group: KRAS_EX2F/R2 μ mol/L, EGFR_EX19F/R1 μ mol/L, EGFR_EX20F/R0.5 μ mol/L, NRAS_EX2F/R1 μ mol/L, NRAS_EX3F/R1 μ mol/L, PIK3CA_EX9F/R1 μ mol/L;
P2 group: EGFR_EX19F/R0.5 μ mol/L, NRAS_EX2F/R0.5 μ mol/L, BRAF_EX15F/R0.5 μ mol/L, NRAS_EX3F/R0.5 μ mol/L, PIK3CA_EX20F/R1 μ mol/L, MEK1_EX2F/R1;
P3 group: KRAS_EX2F/R2 μ mol/L, EGFR_EX19F/R0.5 μ mol/L, EGFR_EX21F/R0.5 μ mol/L, BRAF_EX15F/R0.5 μ mol/L, PIK3CA_EX9F/R1 μ mol/L, NRAS_EX2F/R0.5 μ mol/L;
P4 group: MEK1_EX2F/R0.5 μ mol/L, PIK3CA_EX20F/R1 μ mol/L, EGFR_EX19F/R0.5 μ mol/L, PTEN_EX6F/R0.5 μ mol/L, EGFR_EX18F/R0.5 μ mol/L;
P5 group: EGFR_EX21F/R0.5 μ mol/L, KRAS_EX2F/R1 μ mol/L, MEK1_EX2F/R0.5 μ mol/L, EGFR_EX18F/R0.5 μ mol/L, BRAF_EX11F/R0.5 μ mol/L, PIK3CA_EX20F/R0.5 μ mol/L, NRAS_EX3F/R0.5 μ mol/L, BRAF_EX15F/R0.5 μ mol/L;
P6 group: NRAS_EX2F/R2 μ mol/L, KRAS_EX2F/R1 μ mol/L, PTEN_EX5F/R1 μ mol/L, PTEN_EX7F/R2 μ mol/L, PIK3CA_EX9F/R1 μ mol/L, BRAF_EX11F/R1 μ mol/L;
Use respectively amplimer mixed solution P1, P2, P3, P4, P5, P6 to carry out the multiplex PCR amplification, obtain the PCR product;
(3) PCR product digestion: the PCR product that step (2) obtains is through alkaline phosphatase and exonuclease digestion; The digestion system is: PCR product 2 μ l, exonuclease 0.5 μ l, alkaline phosphatase 1 μ l, H
2O6.5 μ l; Digestion condition is: 37 ℃ 60 minutes, 75 ℃ 15 minutes;
(4) be designed for the extension primer that detects hot mutant site, specific as follows:
BRAF1397_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGGGACAAAGAATTGGATCTG-3'；
BRAF1406_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTGGAATTGGATCTGGATCATTTG-3'；
BRAF1789_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACTCAGTAAAAATAGGTGATTTTGGT-3'；
BRAF1798_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTCTGACTGGTGATTTTGGTCTAGCTACA-3'；
BRAF1799_extF：5'-TTTTTTTTTTTTTTCTGACTGACTGTGATTTTGGTCTAGCTACAG-3'；
EGFR_2156_ext：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACTGACTGACTCAAAAAGATCAAAGTGCTGG-3'；
EGFR_2582_extR：5'-TTCTCTTCCGCACCCAGC-3'；
EGFR2155_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTCAAAAAGATCAAAGTGCTG-3'；
EGFR2235_2249del_extF：5'-TTTTTTTTTTTTTTTTTTGTTCCCGTCGCTATCAA-3'；
EGFR2235_2249del_extR：5'-TGGCTTTCGGAGATGTT-3'；
EGFR2236_2250del_extF：5'-TTTTTTTTTTTTTTTTTTGTCCCGTCGCTATCAAG-3'；
EGFR2236_2250del_extR：5'-TTTTTTTTTTTTTTTTTTTTGGCTTTCGGAGATGT-3'；
EGFR2369_extR：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTGACTAAGGGCATGAGCTGC-3'；
EGFR2573_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTGACAGATCACAGATTTTGGGC-3'；
KRAS34_extR：5'-TTTTTACTGCTCTTGCCTACGCCAC-3'；
KRAS35_extF：5'-TTTTTTCTTGTGGTAGTTGGAGCTG-3'；
KRAS37_extF：5'-TTTTTTTTGATGGTAGTTGGAGCTGGT-3'；
KRAS38_extF：5'-TTTTTTTTTTTGGTAGTTGGAGCTGGTG-3'；
MEK1_167_extF：5'-TTTTTTTTGACTGACTCTTGAGGCCTTTCTTACCC-3'；
MEK1_171_extF：5'-GGCCTTTCTTACCCAGAA-3'；
MEK1_199_extR：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT?TTTCTGACTCTCACTGATCTTCTCAAAGT-3'；
NRAS181_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACACATACTGGATACAGCTGGA-3'；
NRAS182_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATACTGGATACAGCTGGAC-3'；
NRAS183_extR：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACTGACTGCTCATGGCACTGTACTCTTC-3'；
NRAS34：5'-TTTTTTTTTTTTTTTGGTTGGAGCA-3'；
NRAS35_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGTGGTGGTTGGAGCAG-3'；
NRAS37_extR：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGCTTTTCCCAACAC-3'；
NRAS38_extF：5'-TTTTTTTTTTTTACTGGTGGTGGTTGGAGCAGGTG-3'；
PIK3CA1624_extR：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTGACTTCTCCTGCTCAGTGATTT-3'；
PIK3CA1633_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGATCCTCTCTCTGAAATCACT-3'；
PIK3CA1636_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACTGCCTCTCTCTGAAATCACTGAG-3'；
PIK3CA1637_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACTGCTCTCTCTGAAATCACTGAGC-3'；
PIK3CA3139_extR：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACTGACTGACGTCCAGCCACCATGAT-3'；
PIK3CA3140_extR：5'-GTCCAGCCACCATGA-3'；
PIK3CA3145_extR：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTGATGACTGACTGACTGATTTTGTTGTCCAGCCAC-3'；
PTEN388_extF：5'-TTTTTTTTTCTGACTTGTAAAGCTGGAAAGGGA-3'；
PTEN517_extF：5'-TTTTTTTTTTTTTTTTTTTTTTAGTAACTATTCCCAGTCAGAGG-3'；
PTEN697_extR：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTGAACTTGTCTTCCCGTC-3'；
PTEN800_extF：5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAACAGAACAAGATGCTAAAAA-3'；
(5) extension: the extension primer of step (4) is mixed with the primer mixed solution, is 6 groups, specific as follows:
E1 group: KRAS34_extR9pmol/L, EGFR2235_2249extF5pmol/L, EGFR2369_extR12pmol/L, NRAS37_extF3pmol/L, NRAS181_extF15pmol/L, PIK3CA1633_extF3pmol/L;
E2 group: EGFR2235_2249extR1pmol/L, NRAS38_extR14pmol/L, BRAF1799_extF3pmol/L, NRAS182_extF3pmol/L, MEK1_199_extR12pmol/L;
E3 group: KRAS35_extR1.3pmol/L, EGFR2236_2250extF3.1pmol/L, EGFR2573_extF7pmol/L, BRAF1798_extF2pmol/L, PIK3CA1624_extR6.5pmol/L, PIK3CA1637_extF2.5pmol/L, NRAS35_extF3pmol/L;
E4 group: MEK1_171_extR5pmol/L, PIK3CA3140_extR1.8pmol/L, EGFR2236_2250extR5.5pmol/L, PTEN517_extF8.5pmol/L, EGFR_2156_ext10pmol/L, PIK3CA3139_extR5.5pmol/L;
E5 group: EGFR_2582_extR1pmol/L, KRAS37_extF1pmol/L, MEK1_167_extF2pmol/L, EGFR2155_extF4pmol/L, BRAF1406_extF9pmol/L, PIK3CA3145_extR12.5pmol/L, NRAS183_extR12.5pmol/L, BRAF1789_extF11pmol/L;
E6 group: NRAS34_extR3pmol/L, KRAS38_extF2pmol/L, PTEN388_extF13pmol/L, PTEN697_extR5pmol/L, PTEN800_extF14pmol/L, PIK3CA1636_extF12pmol/L, BRAF1397_extF12.3pmol/L;
The SNaPshot Multiplex Ready Reaction Mix enzyme that adopts AB company to produce carries out respectively extension to the postdigestive PCR product of step (3);
(6) enzymic digestion: in the extension product, add alkaline phosphatase and digest;
(7) electrophoresis: postdigestive extension products is got 1 μ l, adds 9 μ l methane amides, 0.2 μ l LIZ120, mixing, carry out capillary electrophoresis at AB3730 genetic analysis instrument, electrophoresis result draws the sudden change situation of each goal gene with the GeneMapper4.1 software analysis, makes the result and judges;
(8) result judges: 1) according to the location positioning gene locus at electrophoresis peak; 2) determine the base type according to the color at electrophoresis peak: blueness represents the G base, green represents the A base, redness represents the T base, black represents the C base.
2. detection nonsmall-cell lung cancer according to claim 1 drives the method for gene mutation spectrum, it is characterized in that: the condition of the described multiplex PCR amplification of step (2) is: 95 ℃ 5 minutes; (95 ℃ 20 seconds, 58 ℃ 30 seconds, 72 ℃ 1 minute), 40 circulations; 72 ℃ 3 minutes;
The amplification system of the described multiplex PCR of step (2) is: Premix Hotstart Taq5 μ l, amplimer mixed solution 1 μ l, dna profiling 20ng, distilled water is mended to 10 μ l.
3. detection nonsmall-cell lung cancer according to claim 1 drives the method for gene mutation spectrum, and it is characterized in that: the described extension system of step (5) is: SNaPshot Multiplex Ready Reaction Mix2 μ l, postdigestive PCR product 2 μ l, extension primer mixed solution 1 μ l, H
2O5 μ l;
The described extension condition of step (5) is: 96 ℃ 30 seconds; (96 ℃ 10 seconds, 50 ℃ 10 seconds, 60 ℃ 30 seconds), 35 circulations;
The condition of the described enzymic digestion of step (6) is: extension product 9 μ l, alkaline phosphatase 1 μ l; 37 ℃ of digestion in 60 minutes, 75 ℃ of 15 minutes remaining enzymes of deactivation.
4. detection nonsmall-cell lung cancer according to claim 1 drives the method for gene mutation spectrum, and it is characterized in that: the operational condition of the described capillary electrophoresis of step (7) is AB3730 genetic analysis instrument user manual Standard operation procedure SOP.
5. one kind is detected the test kit that nonsmall-cell lung cancer drives gene mutation spectrum, it is characterized in that: the amplimer and the described extension primer for detection of hot mutant site that comprise seven kinds of genes involved exons of the amplification nonsmall-cell lung cancer section described in the claim 1.
6. detection nonsmall-cell lung cancer according to claim 5 drives the test kit of gene mutation spectrum, it is characterized in that: comprise the amplimer mixed solution and extend the primer mixed solution;
Described amplimer mixed solution is as follows:
P1 group: KRAS_EX2F/R2 μ mol/L, EGFR_EX19F/R1 μ mol/L, EGFR_EX20F/R0.5 μ mol/L, NRAS_EX2F/R1 μ mol/L, NRAS_EX3F/R1 μ mol/L, PIK3CA_EX9F/R1 μ mol/L;
P2 group: EGFR_EX19F/R0.5 μ mol/L, NRAS_EX2F/R0.5 μ mol/L, BRAF_EX15F/R0.5 μ mol/L, NRAS_EX3F/R0.5 μ mol/L, PIK3CA_EX20F/R1 μ mol/L, MEK1_EX2F/R1;
P3 group: KRAS_EX2F/R2 μ mol/L, EGFR_EX19F/R0.5 μ mol/L, EGFR_EX21F/R0.5 μ mol/L, BRAF_EX15F/R0.5 μ mol/L, PIK3CA_EX9F/R1 μ mol/L, NRAS_EX2F/R0.5 μ mol/L;
P4 group: MEK1_EX2F/R0.5 μ mol/L, PIK3CA_EX20F/R1 μ mol/L, EGFR_EX19F/R0.5 μ mol/L, PTEN_EX6F/R0.5 μ mol/L, EGFR_EX18F/R0.5 μ mol/L;
P5 group: EGFR_EX21F/R0.5 μ mol/L, KRAS_EX2F/R1 μ mol/L, MEK1_EX2F/R0.5 μ mol/L, EGFR_EX18F/R0.5 μ mol/L, BRAF_EX11F/R0.5 μ mol/L, PIK3CA_EX20F/R0.5 μ mol/L, NRAS_EX3F/R0.5 μ mol/L, BRAF_EX15F/R0.5 μ mol/L;
P6 group: NRAS_EX2F/R2 μ mol/L, KRAS_EX2F/R1 μ mol/L, PTEN_EX5F/R1 μ mol/L, PTEN_EX7F/R2 μ mol/L, PIK3CA_EX9F/R1 μ mol/L, BRAF_EX11F/R1 μ mol/L;
Described extension primer mixed solution is as follows:
E1 group: KRAS34_extR9pmol/L, EGFR2235_2249extF5pmol/L, EGFR2369_extR12pmol/L, NRAS37_extF3pmol/L, NRAS181_extF15pmol/L, PIK3CA1633_extF3pmol/L;
E2 group: EGFR2235_2249extR1pmol/L, NRAS38_extR14pmol/L, BRAF1799_extF3pmol/L, NRAS182_extF3pmol/L, MEK1_199_extR12pmol/L;
E3 group: KRAS35_extR1.3pmol/L, EGFR2236_2250extF3.1pmol/L, EGFR2573_extF7pmol/L, BRAF1798_extF2pmol/L, PIK3CA1624_extR6.5pmol/L, PIK3CA1637_extF2.5pmol/L, NRAS35_extF3pmol/L;
E4 group: MEK1_171_extR5pmol/L, PIK3CA3140_extR1.8pmol/L, EGFR2236_2250extR5.5pmol/L, PTEN517_extF8.5pmol/L, EGFR_2156_ext10pmol/L, PIK3CA3139_extR5.5pmol/L;
E5 group: EGFR_2582_extR1pmol/L, KRAS37_extF1pmol/L, MEK1_167_extF2pmol/L, EGFR2155_extF4pmol/L, BRAF1406_extF9pmol/L, PIK3CA3145_extR12.5pmol/L, NRAS183_extR12.5pmol/L, BRAF1789_extF11pmol/L;
E6 group: NRAS34_extR3pmol/L, KRAS38_extF2pmol/L, PTEN388_extF13pmol/L, PTEN697_extR5pmol/L, PTEN800_extF14pmol/L, PIK3CA1636_extF12pmol/L, BRAF1397_extF12.3pmol/L.
According to claim 5 or 6 described detection nonsmall-cell lung cancers drive the test kit of gene mutation spectrums, it is characterized in that: also comprise Premix Hotstart Taq, alkaline phosphatase and exonuclease and SNaPshot Multiplex Ready Reaction Mix enzyme.
8. detection nonsmall-cell lung cancer claimed in claim 7 drives the application of the test kit of gene mutation spectrum, it is characterized in that: described detection nonsmall-cell lung cancer drives the test kit of gene mutation spectrum and uses in clinical as diagnostic reagent.
9. detection nonsmall-cell lung cancer according to claim 8 drives the application of the test kit of gene mutation spectrum, it is characterized in that may further comprise the steps:
1. carry out respectively pcr amplification with amplimer mixed solution P1, P2, P3, P4, P5, P6, obtain the PCR product; The pcr amplification condition is: 95 ℃ 5 minutes; (95 ℃ 20 seconds, 58 ℃ 30 seconds, 72 ℃ 1 minute), 40 circulations; 72 ℃ 3 minutes; The pcr amplification system is: Premix Hotstart Taq5 μ l, amplimer mixed solution 1 μ l, dna profiling 20ng, and distilled water is mended to 10 μ l;
2. the PCR product that 1. step is obtained is through alkaline phosphatase and exonuclease digestion; The digestion system is: PCR product 2 μ l, exonuclease 0.5 μ l, alkaline phosphatase 1 μ l, H
2O6.5 μ l; Digestion condition is: 37 ℃ 60 minutes, 75 ℃ 15 minutes;
3. extension: use and extend primer mixed solution E1, E2, E3, E4, E5 and E6 and carry out respectively extension; The extension system is: SNaPshot Multiplex Ready Reaction Mix2 μ l, PCR product 2 μ l, extension primer mixed solution 1 μ l, H
2O5 μ l; The extension condition is: 96 ℃ 30 seconds; (96 ℃ 10 seconds, 50 ℃ 10 seconds, 60 ℃ 30 seconds), 35 circulations;
4. the alkaline phosphatase that adds 1 μ l in the every pipe PCR product that 3. step is obtained, 37 ℃ of digestion in 60 minutes, 75 ℃ of 15 minutes remaining enzymes of deactivation;
5. postdigestive extension products is got 1 μ l, adds 9 μ l methane amides, 0.2 μ l LIZ120, and mixing carries out capillary electrophoresis at AB3730 genetic analysis instrument; Electrophoresis result draws the sudden change situation of each goal gene with the GeneMapper4.1 software analysis, make the result and judge.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310284338.XA CN103305625B (en) | 2013-07-08 | 2013-07-08 | Method and kit for detecting non-small cell lung cancer drive gene mutation spectrum, and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310284338.XA CN103305625B (en) | 2013-07-08 | 2013-07-08 | Method and kit for detecting non-small cell lung cancer drive gene mutation spectrum, and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103305625A true CN103305625A (en) | 2013-09-18 |
| CN103305625B CN103305625B (en) | 2014-06-25 |
Family
ID=49131341
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310284338.XA Active CN103305625B (en) | 2013-07-08 | 2013-07-08 | Method and kit for detecting non-small cell lung cancer drive gene mutation spectrum, and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103305625B (en) |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232750A (en) * | 2014-05-30 | 2014-12-24 | 嘉兴雅康博医学检验所有限公司 | NRAS gene mutation detection kit |
| CN104450948A (en) * | 2014-12-31 | 2015-03-25 | 北京圣谷同创科技发展有限公司 | Cancer detecting method, kit and application thereof |
| CN104732116A (en) * | 2015-03-13 | 2015-06-24 | 西安交通大学 | Method for screening cancer driver gene based on biological network |
| CN104762410A (en) * | 2015-04-29 | 2015-07-08 | 上海允英医疗科技有限公司 | Primer, probes and detection kit used for full RAS mutation detection |
| CN105567854A (en) * | 2016-03-01 | 2016-05-11 | 济南银丰医学检验有限公司 | Primer combination for detecting human EGFR, KRAS and BRAF gene mutation and kit thereof |
| CN105813643A (en) * | 2013-11-27 | 2016-07-27 | 翁科埃斯克斯有限公司 | Method of treating non-small-cell lung cancer using pharmaceutical formulation containing thienotriazolodiazepine compounds |
| CN106544427A (en) * | 2016-10-27 | 2017-03-29 | 广东省人民医院(广东省医学科学院) | A kind of test kit in detection pulmonary carcinoma EGFR genetic mutation targeted therapy correlation Resistance mutation site |
| CN106636442A (en) * | 2017-02-23 | 2017-05-10 | 上海鼎晶生物医药科技股份有限公司 | Combined detection kit for human tumor gene mutation |
| CN106676105A (en) * | 2015-11-04 | 2017-05-17 | 深圳市瀚海基因生物科技有限公司 | Primers capable of simultaneously acquiring and detecting multiple target area sequences, kit and method |
| CN106709278A (en) * | 2017-01-10 | 2017-05-24 | 河南省医药科学研究院 | Method for carrying out screening and functional analysis on driver genes of NSCLC (Non-Small Cell Lung Cancer) |
| CN106755400A (en) * | 2016-12-21 | 2017-05-31 | 上海浦美生物医药科技有限公司 | A kind of detection method of blood EGFR gene T790M mutation |
| WO2017181134A3 (en) * | 2016-04-15 | 2017-12-21 | F. Hoffman-La Roche Ag | Detecting cancer driver genes and pathways |
| CN107513577A (en) * | 2017-10-19 | 2017-12-26 | 南京科维思生物科技股份有限公司 | A kind of method of efficient detection EGFRT790M mutant and probe and kit for detection |
| CN108179191A (en) * | 2018-02-05 | 2018-06-19 | 广州市达瑞生物技术股份有限公司 | A kind of kit for detecting mankind's ctDNA gene mutations |
| CN108410982A (en) * | 2018-02-05 | 2018-08-17 | 广州市达瑞生物技术股份有限公司 | A kind of kit based on 34 mutational sites of MALDI-TOF-MS detection lung cancer |
| CN109266744A (en) * | 2018-09-14 | 2019-01-25 | 广州奇辉生物科技有限公司 | Multiple PCR primer, kit and the method for targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology |
| CN109517899A (en) * | 2018-12-05 | 2019-03-26 | 山东普瑞斯分子诊断技术有限责任公司 | For detecting the kit and detection method of EGFR genetic mutation |
| CN109825590A (en) * | 2019-04-01 | 2019-05-31 | 常州桐树生物科技有限公司 | Method, kit and the primer of tumour driving gene mutation and microsatellite instability joint-detection |
| CN114622003A (en) * | 2020-12-11 | 2022-06-14 | 上海吉凯医学检验所有限公司 | Lung cancer related gene mutation detection kit suitable for MassARRAY mass spectrometry platform Iplex analysis |
-
2013
- 2013-07-08 CN CN201310284338.XA patent/CN103305625B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| MARIO TURRI–ZANONI MD ET AL.: "Sinonasal mucosal melanoma: Molecular profile and therapeutic implications from a series of 32 cases", 《HEAD&NECK》 * |
| PIOTR WÓJCIK ET AL.: "BRAF MUTATIONS IN SPORADIC COLORECTAL CARCINOMA FROM POLISH PATIENTS", 《PLO J PATHOL》 * |
| ZENGLIU SU ET AL.: "A Platform for Rapid Detection of Multiple Oncogenic Mutations With Relevance to Targeted Therapy in Non–Small-Cell Lung Cancer", 《THE JOURNAL OF MOLECULAR DIAGNOSTICS》 * |
Cited By (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105813643A (en) * | 2013-11-27 | 2016-07-27 | 翁科埃斯克斯有限公司 | Method of treating non-small-cell lung cancer using pharmaceutical formulation containing thienotriazolodiazepine compounds |
| CN104232750A (en) * | 2014-05-30 | 2014-12-24 | 嘉兴雅康博医学检验所有限公司 | NRAS gene mutation detection kit |
| CN104450948A (en) * | 2014-12-31 | 2015-03-25 | 北京圣谷同创科技发展有限公司 | Cancer detecting method, kit and application thereof |
| CN104450948B (en) * | 2014-12-31 | 2018-03-09 | 北京圣谷同创科技发展有限公司 | Method for detecting cancer, kit and its application |
| CN104732116B (en) * | 2015-03-13 | 2017-11-28 | 西安交通大学 | A kind of screening technique of the cancer driving gene based on bio-networks |
| CN104732116A (en) * | 2015-03-13 | 2015-06-24 | 西安交通大学 | Method for screening cancer driver gene based on biological network |
| CN104762410A (en) * | 2015-04-29 | 2015-07-08 | 上海允英医疗科技有限公司 | Primer, probes and detection kit used for full RAS mutation detection |
| CN106676105A (en) * | 2015-11-04 | 2017-05-17 | 深圳市瀚海基因生物科技有限公司 | Primers capable of simultaneously acquiring and detecting multiple target area sequences, kit and method |
| CN105567854A (en) * | 2016-03-01 | 2016-05-11 | 济南银丰医学检验有限公司 | Primer combination for detecting human EGFR, KRAS and BRAF gene mutation and kit thereof |
| WO2017181134A3 (en) * | 2016-04-15 | 2017-12-21 | F. Hoffman-La Roche Ag | Detecting cancer driver genes and pathways |
| CN106544427A (en) * | 2016-10-27 | 2017-03-29 | 广东省人民医院(广东省医学科学院) | A kind of test kit in detection pulmonary carcinoma EGFR genetic mutation targeted therapy correlation Resistance mutation site |
| CN106755400A (en) * | 2016-12-21 | 2017-05-31 | 上海浦美生物医药科技有限公司 | A kind of detection method of blood EGFR gene T790M mutation |
| CN106709278B (en) * | 2017-01-10 | 2019-05-28 | 河南省医药科学研究院 | A kind of method of non-small cell lung cancer driving genescreen and functional analysis |
| CN106709278A (en) * | 2017-01-10 | 2017-05-24 | 河南省医药科学研究院 | Method for carrying out screening and functional analysis on driver genes of NSCLC (Non-Small Cell Lung Cancer) |
| CN106636442A (en) * | 2017-02-23 | 2017-05-10 | 上海鼎晶生物医药科技股份有限公司 | Combined detection kit for human tumor gene mutation |
| CN107513577A (en) * | 2017-10-19 | 2017-12-26 | 南京科维思生物科技股份有限公司 | A kind of method of efficient detection EGFRT790M mutant and probe and kit for detection |
| CN108179191A (en) * | 2018-02-05 | 2018-06-19 | 广州市达瑞生物技术股份有限公司 | A kind of kit for detecting mankind's ctDNA gene mutations |
| CN108410982A (en) * | 2018-02-05 | 2018-08-17 | 广州市达瑞生物技术股份有限公司 | A kind of kit based on 34 mutational sites of MALDI-TOF-MS detection lung cancer |
| CN109266744A (en) * | 2018-09-14 | 2019-01-25 | 广州奇辉生物科技有限公司 | Multiple PCR primer, kit and the method for targeting sequencing detection lung cancer gene based on UMI unimolecule label noise reduction technology |
| CN109517899A (en) * | 2018-12-05 | 2019-03-26 | 山东普瑞斯分子诊断技术有限责任公司 | For detecting the kit and detection method of EGFR genetic mutation |
| CN109517899B (en) * | 2018-12-05 | 2021-07-20 | 山东普瑞斯分子诊断技术有限责任公司 | Kit for detecting EGFR gene mutation and detection method |
| CN109825590A (en) * | 2019-04-01 | 2019-05-31 | 常州桐树生物科技有限公司 | Method, kit and the primer of tumour driving gene mutation and microsatellite instability joint-detection |
| CN109825590B (en) * | 2019-04-01 | 2022-08-05 | 常州桐树生物科技有限公司 | Method, kit and primer for joint detection of tumor-driven gene mutation and microsatellite instability |
| CN114622003A (en) * | 2020-12-11 | 2022-06-14 | 上海吉凯医学检验所有限公司 | Lung cancer related gene mutation detection kit suitable for MassARRAY mass spectrometry platform Iplex analysis |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103305625B (en) | 2014-06-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103305625B (en) | Method and kit for detecting non-small cell lung cancer drive gene mutation spectrum, and application | |
| CN104818320B (en) | Primer, probe, detection architecture and the kit of disposable detection lung cancer multiple gene | |
| CN104946739B (en) | EGFR genetic mutation detection kit and its application | |
| CN104818318B (en) | Primer, probe, detection architecture and the kit of disposable detection lung cancer multiple gene | |
| CN104372103B (en) | NRAS gene mutation detection kit | |
| CN104894269B (en) | Based on the lung cancer gene profile detection kit that MassARRAY mass spectrometric platforms Iplex analyzes | |
| CN106497920A (en) | A kind of library constructing method and test kit for nonsmall-cell lung cancer detection in Gene Mutation | |
| CN101092644B (en) | Quick detecting gene mutation correlative to curative effect of non small-cell carcinoma of the lung | |
| Song et al. | Clinicopathologic characteristics, genetic variability and therapeutic options of RET rearrangements patients in lung adenocarcinoma | |
| CN111235272B (en) | Composition for once detecting multiple gene mutation of lung cancer and application thereof | |
| CN104450948A (en) | Cancer detecting method, kit and application thereof | |
| CN105567854A (en) | Primer combination for detecting human EGFR, KRAS and BRAF gene mutation and kit thereof | |
| Liu et al. | Potential clinical significance of plasma-based KRAS mutation analysis using the COLD-PCR/TaqMan®-MGB probe genotyping method | |
| CN110938693A (en) | Primer group, kit and method for detecting BRAF gene mutation | |
| CN107513578A (en) | A kind of nucleic acid Mass Spectrometry detection method early sieved for lung cancer driving gene and tumor susceptibility gene | |
| Cheng et al. | Clinical validation of a cell-free DNA gene panel | |
| CN106480201A (en) | Metastasis in Breast Cancer assesses test kit | |
| CN108220444A (en) | A kind of primer combination of probe of KRAS gene mutation detection and its application | |
| CN112029865A (en) | Probe group and gene chip for simultaneously detecting multiple gene mutation types and application thereof | |
| CN105803076A (en) | Specific primer and probe for detecting T790M locus of EGFR gene | |
| Li et al. | The cornerstone of integrating circulating tumor DNA into cancer management | |
| CN109182529B (en) | Specific probe and kit for detecting T790M, C797S and L798I sites of EGFR gene | |
| Zhao et al. | A novel method for detection of mutation in epidermal growth factor receptor | |
| CN110373453A (en) | A kind of detection primer, probe and the kit in KRAS gene mutation site | |
| Wang et al. | Universal and highly accurate detection of circulating tumor DNA mutation in non-small cell lung cancer based on CRISPR/Cas12a system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |