CN108410982A

CN108410982A - A kind of kit based on 34 mutational sites of MALDI-TOF-MS detection lung cancer

Info

Publication number: CN108410982A
Application number: CN201810112790.0A
Authority: CN
Inventors: 杨学习; 吴英松; 李明; 朱安娜; 李晓敏; 蔡晶晶
Original assignee: Guangzhou Da Rui Biotechnology Ltd
Current assignee: Guangzhou Da Rui Biotechnology Ltd
Priority date: 2018-02-05
Filing date: 2018-02-05
Publication date: 2018-08-17

Abstract

The invention discloses a kind of kits based on 34 mutational sites of MALDI TOF MS detection lung cancer, which includes that multiplexed PCR amplification reacts primer, such as SEQ ID NO：Shown in 1~16；And Single base extension primer, such as SEQ ID NO：Shown in 17~48.The present invention can detect 3 sites in 23 sites of EGFR gene, 7 sites of KRAS genes, 1 site of BRAF and PIK3CA genes simultaneously.By the invention it is possible to which 34 sites of 4 genes are carried out to carry out Single base extension in target fragment amplification and 8 holes in 2 holes, the present invention can be carried out at the same time detection and analysis to 34 site mutations in this 4 genes that may be present in exceptional sample.Kit through the invention understands correlative factor of the patient to drug susceptibility, carries out individualized treatment for patients with lung cancer and targeted drug is applicable in important directive significance.

Description

A kind of kit based on 34 mutational sites of MALDI-TOF-MS detection lung cancer

Technical field

The present invention relates to lung cancer mutational site detection technique fields, and MALDI-TOF-MS is based on more particularly, to one kind Detect the kit in 34 mutational sites of lung cancer.

Background technology

Lung cancer is that one of most common malignant tumour in the world today, tumor incidence and lethality top the list in male Position, is only second to breast cancer in women.Chemotherapy is to treat the main means of middle and advanced stage lung cancer and patient is preoperative, art at present One of the method for auxiliary treatment afterwards.Clinically some patientss chemotherapy effect is not good enough, chemotherapy failure, the main reason is that tumour is thin Born of the same parents are insensitive to anticancer drug or generate drug resistance.Therefore, understand correlative factor of the patient to drug susceptibility, lung cancer is suffered from Person carries out individualized treatment and targeted drug is applicable in guidance and is particularly important.

Clinical discovery EGFR genetic mutation carries EGFR with having correlation, the overwhelming majority the effect of NSCLC targeted therapies The patient of gene mutation is significant in efficacy.Numerous studies data shows that EGFR genetic mutation is concentrated mainly on tyrosine kinase area On (tyrosine kinase coding domain) exons 1 8-21, wherein 19 exons are mostly in-frame deletion (746- 753) property is mutated, and accounts for about the 45% of all mutation；21 exons are mostly to substitute mutation (mainly L858R), account for about all mutation 40%.At present it is believed that the two hot spot mutations can enhance sensibility of the tumour cell to TKIs, and can be used as Index is effectively predicted in TKIs treatments.Therefore, detection EGFR genetic mutation is for instructing NSCLC patients clinical medications to have weight The reference value wanted.

There are three types of RAS gene families and the relevant gene of human tumor --- and HRAS, KRAS and NRAS are respectively positioned at 11, on 12 and No. 1 chromosomes.In RAS genes, KRAS influences human cancer maximum.When normal, it is thin that it can control regulation and control The path of intracellular growth；When being abnormal, then lead to cell continued propagation, and prevent cell self-destruction, cannot generate normal RAS albumen keeps Cellular Signaling Transduction Mediated disorderly, uncontrolled cellular proliferation and canceration.KRAS mutation betides kinds cancer, such as Lung cancer, colon cancer etc..90% mutation is happened at 12,13 bit codons, can lead to the different of the growth signals of p21-ras albumen Often variation.The gene is the target molecule of TKIs treatments, and the patient for carrying KRAS gene mutation is insensitive to TKIs treatments.It crosses The growth signals of degree can stimulating cellular growth and proliferation so as to cause cancer generation.

A large amount of clinical researches show that targeted drug can reach 60% to patient's effective percentage that KRAS gene mutation does not occur, And it is then completely ineffective to the patient that KRAS gene mutation has occurred.KRAS genetic tests are that current doctor understands patient oncogene Situation most directly, most efficient method.By detecting KRAS genes either with or without mutation, anti-EGFR (epidermal growths can be filtered out Factor acceptor) the effective PATIENTS WITH LARGE BOWEL of targeted drug treatment, the individualized treatment of tumour patient is realized, to extend patient Life cycle.For the patient not being mutated, then unnecessary medical expense and toxic side effect can be reduced.

BRAF is a kind of oncogene, it encodes a kind of serine/threonine specificity kinase, RAS/RAF/MEK/ ERK/MAPK The important transduced element of access participates in various biological event in regulating cell, such as cell growth, differentiation and apoptosis.Research Show in a variety of human malignancies, such as lung cancer, malignant mela noma, colorectal cancer have the BRAF of different proportion Gene mutation.The BRAF gene mutation of about 80-90% is happened on 1799 nucleotide of exon15, and T sports A, leads to it The glutamic acid of coding replaces (V600E) by valine.It is now recognized that V600E mutation can simulate two sites T599 and S602 Phosphorylation events, to make BRAF abnormal proteins activate.Generation, development and the clinical knot of BRAF mutation status and kinds of tumors Fruit is related.

Clinical studies show, BRAF gene mutation patient to EGFR targeted drugs there may be initial drug-resistant, therefore NCCN It is recommended that being using targeted drug, reply KRAS gene wild type patients further detect BRAF gene state.BRAF gene Abrupt climatic change can improve the specific aim of clinical treatment, instruct the reasonable employment of targeted drug, and invalid or malpractice is avoided to cause Patient's state of an illness delay, reduce Operative risk.

PIK3CA genes are phosphatidylinositol 3-kinase catalytic subunit α genes, are 3q26. 3 in the position of chromosome, compile The p110 α catalytic subunits of code Ι type phosphatidylinositol-3-kinases.Phosphatidylinositol-3-kinase/serine-serine/threonine protein kinase Enzyme B (PI3K-Akt) signal path is the signal of interest approach for adjusting cell function, in critically important in Cellular signalling network Position, it is related to transmitter loss etc. with the rearrangement of the proliferation of cell, adherency, differentiation, cytoskeleton structure.PI3K conducts EGFR downstream signaling molecules are activated, and tumour cell is caused drug resistance occur to drugs such as EGFR-TKI.So detection PIK3CA Gene mutation can predict drug resistance of the patients with lung cancer to drugs such as EGFR-TKI, for instructing the targeting medication of patient to have Significance.

There are many method of detection gene mutation both at home and abroad, such as generation PCR sequencing PCR, single-strand conformation polymorphism analysis method, limitation Property fragment length polymorphism analysis method, two generation sequencing approaches, cut model and flashlight model detector etc..But these methods are due to list The gene and bit number of points of secondary detection are limited or the later stage needs special analysis of biological information, and there is of high cost, operation is multiple Miscellaneous, the problems such as time-consuming, is unfavorable for Big Clinical Samples detection.Therefore, find and establish a low cost, high throughput, simplicity easily Row, quick and precisely reliable detection method, are generalized to clinical application, are a urgent problems to be solved.

Invention content

The purpose of the invention is to overcome the deficiencies of the prior art and provide one group for detecting 34 mutation positions of lung cancer The primer combination of point.

It is a further object to provide primer combinations in the reagent for preparing 34 mutational sites of detection lung cancer Application in box.

Third object of the present invention is to provide a kind of examinations based on 34 mutational sites of MALDI-TOF-MS detection lung cancer Agent box.

To achieve the goals above, the present invention is achieved by the following technical programs：

One group of primer combination for detecting 34 mutational sites of lung cancer, primer combination react primer by multiplexed PCR amplification It is formed with Single base extension primer；The sequence such as SEQ ID NO of the multiplexed PCR amplification reaction primer：Shown in 1~16, single alkali The sequence of base extension primer such as SEQ ID NO：Shown in 17~48.

Application of the primer combination as described above in the kit for preparing 34 mutational sites of detection lung cancer.

A kind of kit based on 34 mutational sites of MALDI-TOF-MS detection lung cancer, the kit include as above The primer combination.

Preferably, the detection architecture of the kit handles reaction system and list by multiplexed PCR amplification reaction system, SAP Base extension system forms；Wherein, multiplexed PCR amplification reaction system is by 2 independent multiplexed PCR amplification reaction systems Composition, a multiplexed PCR amplification reaction system use amplimer mixture 1, another multiplexed PCR amplification reaction system to make With amplimer mixture 2；Single base extension system is made of 8 independent single base extension systems, and 8 solely Vertical single base extension system uses extension primer mixture 1,2,3,4,5,6,7,8 respectively；

SEQ ID NO：Multiplexed PCR amplification reaction primer composition amplimer mixture 1 shown in 1~14；

SEQ ID NO：1~6, multiplexed PCR amplification reaction primer composition amplimer shown in 11~12 and 15~16 is mixed Close object 2；

SEQ ID NO：Single base extension primer composition extension primer mixture 1 shown in 17~22；

SEQ ID NO：Single base extension primer composition extension primer mixture 2 shown in 23~26；

SEQ ID NO：Single base extension primer composition extension primer mixture 3 shown in 27~29；

SEQ ID NO：Single base extension primer composition extension primer mixture 4 shown in 30~34；

SEQ ID NO：Single base extension primer composition extension primer mixture 5 shown in 35~38；

SEQ ID NO：Single base extension primer composition extension primer mixture 6 shown in 39~42；

SEQ ID NO：Single base extension primer composition extension primer mixture 7 shown in 43~46；

SEQ ID NO：Single base extension primer composition extension primer mixture 8 shown in 47~48；

Amplimer mixture 1 coordinates extension primer mixture 1~4 to use；

Amplimer mixture 2 coordinates extension primer mixture 5~8 to use.

Preferably, the kit includes multiplexed PCR amplification reaction reagent, SAP reagent treatments, Single base extension examination Agent.

Preferably, multiplexed PCR amplification reaction system is：6.50 μ L purified waters, 2.5 μ L multiplex PCR buffer solutions, 2 μ L chlorinations Magnesium buffer solution, 0.5 μ L dNTP mixtures, 1 μ L multiplex PCR enzymes, 2.5 μ L amplimers mixtures 1 or amplimer mixture 2,10 μ L DNA.

Preferably, the condition of multiplexed PCR amplification reaction is to carry out following steps successively：

(1) 95 DEG C 2 minutes；

(2) 95 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 1 minute, 45 cycle

(3) 72 DEG C 5 minutes；

(4) 4 DEG C of preservations.

Preferably, SAP processing reaction system is：5 μ L multiplexed PCR amplification reaction products, the purifying of 1.53 μ L high pressure sterilizations Water, 0.17 μ L phosphatase buffers, 0.3 μ L phosphoric acid digestion enzymes.

Preferably, SAP processing reaction condition be 37 DEG C 40 minutes, 85 DEG C inactivate 5 minutes.

Preferably, single base extension system is：7 μ L SAP handle reaction product, the purifying of 0.62 μ L high pressure sterilizations Water, 0.20 μ L extend buffer solution, 0.04 μ L catalytic reaction enzymes, and 0.20 μ L extend the one kind terminated in mixed liquor A, T, C, G, One kind in 0.94 μ L extension primers mixture 1~8；

Wherein, extend that terminate mixed liquor A, T, C, G be ddATP, ddTTP, ddCTP and ddGTP these four double deoxidation cores The mixture aqueous solution of thuja acid, wherein the ratio for extending termination mixed liquor A is 1:4:4:4, extending termination mixed liquor T ratios is 4:1:4:4, the ratio for extending termination mixed liquor C is 5:5:1:5, the ratio for extending termination mixed liquor G is 3:3:3:1；

The reacting hole correspondence of extension primer 1,6 and 7 uses extension to terminate mixed liquor G, the reaction of extension primer 2 and 8 Hole, which corresponds to, uses extension to terminate mixed liquor A；The reacting hole correspondence of extension primer 3 and 4 uses extension to terminate mixed liquor The reacting hole correspondence of T, extension primer 5 use extension to terminate mixed liquor C.

Preferably, the condition of single base extension is to carry out following steps successively：

(1) step S1；

(2) step S2；

(3) step S3, step S4, step S5 successively, and recycle 5 times；

(4) (2) and (3) are carried out successively to recycle 40 times；

(5) 4 DEG C of preservations；

Wherein, step S1：95 DEG C 30 seconds, step S2：95 DEG C 5 seconds, step S3：52 DEG C 5 seconds, step S4：80 DEG C 5 seconds, step Rapid S5：72 DEG C 3 seconds.

The multiplex PCR that the present invention is previously mentioned refers to amplified reaction of the multipair primer of mixing in the same reacting hole System；The DNA of sample to be tested is extracted as multiplexed PCR amplification template.According to the present invention, wherein the multiplexed PCR amplification is anti- The recurring number answered is 45 cycles.

The present invention analyzes the site of chromosome specific according to the length of extension products flight time in vacuum tubule Genotype.The SNP site that the present invention selects is the polymorphism for the inhereditary material mononucleotide being present in human body cell, institute The product segment of extension is between 15~30bp.Gene loci is detected using MALDI-TOF-MS technologies, detection extends Flight time of the product in vacuum tubule, the analysis for the loci gene type of chromosome specific provide foundation.

Specific SNP site mutation in EGFR gene of the present invention, selects lot of documents result of study to show In the EGFR gene and relevant site of lung cancer：COSM6239(G719A)、COSM6252 (G719S)、COSM6253 (G719C)、COSM6210(L747-T751>S_2240_2251del12)、 COSM6218(L747-E749del_2239_ 2247delTTAAGAGAA)、COSM6223 (E746-A750del_2235_2249del15)、COSM6255(L747- S752del_2239_2256del18)、 COSM6254(L747-T751del_2239_2253del15)、COSM6225 (E746-A750del_2236-2250del15)、COSM12369 (L747-T751del_2240_2254del15)、 COSM12370 (L747-P753>S_2240_2257del18)、COSM12382 (L747-A750>P_2239_ 2248TTAAGAGAAG>C)、COSM12383 (L747-T751>P_2239_2251>C)、COSM12384(E746-S752>V_ 2237_2255>T)、 COSM12387(L747-P753>Q_2239_2258>CA)COSM12678 (E746-T751>A_2237_ 2251del15)、COSM6240(T790M)、COSM6241(S768I)、 COSM12376(V769_D770insASV)、 COSM12377(H773_V774insH)、COSM12378 (D770_N771insG)、COSM6213(L861Q)、COSM6224 (L858R) catastrophe in this 23 sites SNP.

Specific SNP site mutation on KRAS genes of the present invention, selects lot of documents result of study to show On the KRAS genes and relevant site of lung cancer：COSM516(G12C)、COSM517 (G12S)、COSM518(G12R)、 The mutation feelings of COSM520 (G12V), COSM521 (G12D), COSM522 (G12A), COSM532 (G13D) this 7 SNP sites Condition.

In BRAF gene of the present invention specific SNP site mutation, select literature research result show In the BRAF gene and relevant site of lung cancer：The catastrophe of COSM476 (V600E) this 1 SNP site.

On PIK3CA genes of the present invention specific SNP site mutation, select literature research result show On the PIK3CA genes and relevant site of lung cancer：COSM760(E542K)、COSM763 (E545K)、COSM775(H1047R) The catastrophe of this 3 SNP sites.

The correspondence in the SNP mutation site and primer that are detected see the table below：

Table 1：

Briefly, the present invention includes multiplexed PCR amplification primer, SAP processing and extension primer；Select sample genome DNA is as template；The SNP site of chromosome specific is expanded；Then it uses remaining de- in SAP enzyme-deactivating PCR systems Oxygen ribonucleotide triphosphate (dNTP) and primer；Single base extension is carried out to amplified production with PCR；Using MALDI-TOF-MS Technology carries out molecular weight differentiation according to the length of flight time, and analyzes data.

When mutational site is single base mutation, as soon as MALDI-TOF-MS detections are carried out according to the product of extension primer, It can decide whether to mutate；When insertion or the missing of polybase base occur for mutational site, then according to the need of design of primers Single base extension, the Locus Analysis in Shoots and list of single primer extend are carried out to the one or both ends of insertion or deletion fragment respectively Base mutation it is identical, for both ends extend site need the primer extend result of two Single base extensions is carried out at the same time Analysis, can just judge whether to mutate.Since the mutation of some specific positions is the same position in genome, so Specific position mutation shares identical Single base extension primer.

Likewise, being substantially the same specific position mutation for position, multiple PCR primer can be shared.

Compared with prior art advantage of the invention is that：

1, the present invention can detect simultaneously 23 sites of EGFR gene, 7 sites of KRAS genes, BRAF 1 position 3 sites of point and PIK3CA genes.34 sites of 4 genes can be carried out to target fragment amplification and 8 in 2 holes In hole carry out Single base extension, you can to 34 site mutations in this 4 genes that may be present in exceptional sample simultaneously into Row detection and analysis.

2, sample of the present invention requirement is few, the sensitivity higher of kit, is embodied in each sample PCR and only needs 2 Hole only needs 20ng DNA, one-time detection only to need 40ng DNA per hole.

3, for the present invention in extension, the extension of addition terminates mixed liquor A, T, C, G, this 4 kinds extend termination mixed liquor It is to have carried out what base ratio adjusted according to the base of mutation and wild type, reduces wild type base ratio in reacting hole, extends The variants gone out are more, and peak height becomes apparent from, to improve low frequency sudden change sample recall rate, this method can realize mutation The sample standard deviation that rate is 5% can be detected accurately.

4, extend the use for terminating mixed liquor A, T, C, G so that extension product can directly carry out MALDI-TOF- MS is detected, the processing delayed without resin so that entire detection process is more simple and efficient.

5, sample flux higher is detected, the cost of kit is lower, and operation is more convenient, and automation equipment degree is high.

6, the detection site that the present invention selects, kit through the invention related to the medicaments insensitive of lung cancer understand To the correlative factor of drug susceptibility, patients with lung cancer progress individualized treatment and targeted drug are applicable in be had emphatically patient The directive significance wanted.

Description of the drawings

Fig. 1 is the not mutated detection figure in the sites G719A of EGFR gene

Fig. 2 is the not mutated detection figure in the sites G719S and G719C of EGFR gene.

Fig. 3 is the L747-T751 of EGFR gene>The not mutated detection figure in the sites S_2240_2251del12.

Fig. 4 is the not mutated detection figure in the sites L747-E749del_2239_2247delTTAAGAGAA of EGFR gene.

Fig. 5 is the not mutated detection figure in the sites E746-A750del_2235_2249del15 of EGFR gene.

Fig. 6 is the not mutated detection figure in the sites L747-S752del_2239_2256del18 of EGFR gene.

Fig. 7 is the not mutated detection figure in the sites L747-T751del_2239_2253del15 of EGFR gene.

Fig. 8 is the not mutated detection figure in the sites E746-A750del_2236_2250del15 of EGFR gene.

Fig. 9 is the not mutated detection figure in the sites L747-T751del_2240_2254del15 of EGFR gene.

Figure 10 is the L747-P753 of EGFR gene>The not mutated detection figure in the sites S_2240_2257del18.

Figure 11 is the L747-A750 of EGFR gene>P_2239_2248TTAAGAGAAG>The not mutated detection figure in the sites C.

Figure 12 is the L747-T751 of EGFR gene>P_2239_2251>The not mutated detection figure in the sites C.

Figure 13 is the E746-S752 of EGFR gene>V_2237_2255>The not mutated detection figure in the sites T.

Figure 14 is the L747-P753 of EGFR gene>Q_2239_2258>The not mutated detection figure in the sites CA.

Figure 15 is the E746-T751 of EGFR gene>The not mutated detection figure in the sites A_2237_2251del15.

Figure 16 is the not mutated detection figure in the sites T790M of EGFR gene.

Figure 17 is the not mutated detection figure in the sites S768I of EGFR gene.

Figure 18 is the not mutated detection figure in the sites V769_D770insASV of EGFR gene.

Figure 19 is the not mutated detection figure in the sites H773_V774insH of EGFR gene.

Figure 20 is the not mutated detection figure in the sites D770_N771insG of EGFR gene.

Figure 21 is the not mutated detection figure in the sites L861Q of EGFR gene.

Figure 22 is the not mutated detection figure in the sites L858R of EGFR gene.

Figure 23 is the not mutated detection figure in the site G12C, G12S and G12R of KRAS genes.

Figure 24 is the not mutated detection figure in the site G12V, G12D and G12A of KRAS genes.

Figure 25 is the not mutated detection figure in the sites G13D of KRAS genes.

Figure 26 is the not mutated detection figure in the sites V600E of BRAF gene.

Figure 27 is the not mutated detection figure in the sites E542K of PIK3C genes.

Figure 28 is the not mutated detection figure in the sites E545K of PIK3C genes.

Figure 29 is the not mutated detection figure in the sites H1047R of PIK3C genes.

Figure 30 is the G719A site mutations detection figure of EGFR gene

Figure 31 is the G719S site mutations detection figure of EGFR gene.

Figure 32 is the G719C site mutations detection figure of EGFR gene.

Figure 33 is the L747-T751 of EGFR gene>S_2240_2251del12 site mutations detection figure.

Figure 34 is the L747-E749del_2239_2247delTTAAGAGAA site mutations detection figure of EGFR gene.

Figure 35 is the E746-A750del_2235_2249del15 site mutations detection figure of EGFR gene.

Figure 36 is the L747-S752del_2239_2256del18 site mutations detection figure of EGFR gene.

Figure 37 is the L747-T751del_2239_2253del15 site mutations detection figure of EGFR gene.

Figure 38 is the E746-A750del_2236_2250del15 site mutations detection figure of EGFR gene.

Figure 39 is the L747-T751del_2240_2254del15 site mutations detection figure of EGFR gene.

Figure 40 is the L747-P753 of EGFR gene>S_2240_2257del18 site mutations detection figure.

Figure 41 is the L747-A750 of EGFR gene>P_2239_2248TTAAGAGAAG>C site mutations detection figure.

Figure 42 is the L747-T751 of EGFR gene>P_2239_2251>C site mutations detection figure.

Figure 43 is the E746-S752 of EGFR gene>V_2237_2255>T site mutations detection figure.

Figure 44 is the L747-P753 of EGFR gene>Q_2239_2258>CA site mutations detection figure.

Figure 45 is the E746-T751 of EGFR gene>A_2237_2251del15 site mutations detection figure.

Figure 46 is the T790M site mutations detection figure of EGFR gene.

Figure 47 is the S768I site mutations detection figure of EGFR gene.

Figure 48 is the V769_D770insASV site mutations detection figure of EGFR gene.

Figure 49 is the H773_V774insH site mutations detection figure of EGFR gene.

Figure 50 is the D770_N771insG site mutations detection figure of EGFR gene.

Figure 51 is the L861Q site mutations detection figure of EGFR gene.

Figure 52 is the L858R site mutations detection figure of EGFR gene.

Figure 53 is the G12C site mutations detection figure of KRAS genes.

Figure 54 is the G12S site mutations detection figure of KRAS genes.

Figure 55 is the G12R site mutations detection figure of KRAS genes.

Figure 56 is the G12V site mutations detection figure of KRAS genes.

Figure 57 is the G12D site mutations detection figure of KRAS genes.

Figure 58 is the G12A site mutations detection figure of KRAS genes.

Figure 59 is the G13D site mutations detection figure of KRAS genes.

Figure 60 is the V600E site mutations detection figure of BRAF gene.

Figure 61 is the E542K site mutations detection figure of PIK3C genes.

Figure 62 is the E545K site mutations detection figure of PIK3C genes.

Figure 63 is the H1047R site mutations detection figure of PIK3C genes.

Specific implementation mode

The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the implementation Example is served only for explaining the present invention, is not intended to limit the scope of the present invention.Test method used in following embodiments for example without Specified otherwise is conventional method；Used material, reagent etc. commercially obtain unless otherwise specified Reagent and material.

Embodiment 1

1, a kind of kit based on 34 mutational sites of MALDI-TOF-MS detection lung cancer, the kit include：It is pure Change water, multiplex PCR buffer solution, magnesium chloride buffer solution, dNTP mixtures, multiplex PCR enzyme, amplimer mixture 1~2, phosphoric acid Enzyme buffer liquid, phosphoric acid digestion enzyme extend buffer solution, and base prolongs catalytic reaction enzyme, extends and terminates mixed liquor A, T, C, G, extension primer Mixture 1~8.

Wherein, extend that terminate mixed liquor A, T, C, G be ddATP, ddTTP, ddCTP and ddGTP these four double deoxidation cores The mixture aqueous solution of thuja acid, wherein the ratio for extending termination mixed liquor A is 1:4:4:4, extending termination mixed liquor T ratios is 4:1:4:4, the ratio for extending termination mixed liquor C is 5:5:1:5, the ratio for extending termination mixed liquor G is 3:3:3:1.

2, the use of kit

The detection architecture of the kit is prolonged by multiplexed PCR amplification reaction system, SAP processing reaction systems and single base Stretch reaction system composition.

Multiplexed PCR amplification reaction system is：6.50 μ L purified waters, 2.5 μ L multiplex PCR buffer solutions, 2 μ L magnesium chlorides buffering Liquid, 0.5 μ L dNTP mixtures, 1 μ L multiplex PCR enzymes, 2.5 μ L amplimers mixtures 1 or 2,10 μ L of amplimer mixture DNA；Multiplexed PCR amplification reaction condition is：

SAP handles reaction system：5 μ L multiplexed PCR amplification reaction product A or multiplexed PCR amplification reaction product B are (to expand The product for increasing the progress multiplexed PCR amplification of primer mixture 1 is known as multiplexed PCR amplification reaction product A, with amplimer mixture 2 The product for carrying out multiplexed PCR amplification is known as multiplexed PCR amplification reaction product B), 1.53 μ L high pressure sterilization purified waters, 0.17 μ L phosphorus Sour enzyme buffer liquid, 0.3 μ L phosphoric acid digestion enzymes；SAP handle reaction condition be 37 DEG C 40 minutes, 85 DEG C inactivate 5 minutes.

Single base extension system is：7 μ L SAP processing reaction products A or SAP handle reaction product B (multiplex PCRs Amplified reaction product A carry out SAP treated product be known as SAP processing reaction product A, multiplexed PCR amplification reaction product B into Row SAP treated product is known as SAP processing reaction product B), 0.62 μ L high pressure sterilization purified waters, 0.20 μ L extend buffering Liquid, 0.04 μ L catalytic reaction enzymes, 0.20 μ L, which extend, terminates mixed liquor；Wherein, one kind in A, T, C, G (be added extension primer 1, 6 and 7 reacting hole is accordingly added and extends termination mixed liquor G；The reacting hole of extension primer 2 and 8 is added, is accordingly added and prolongs Stretch termination mixed liquor A；The reacting hole of extension primer 3 and 4 is added, is accordingly added and extends termination mixed liquor T；It is added to extend and draw The reacting hole of object 5, be accordingly added to extend terminate mixed liquor C), one kind in 0.94 μ L extension primers mixture 1~8 is (when adding When entering SAP processing reaction product A, one kind in corresponding extension primer mixture 1~4 handles reaction product B when SAP is added When, correspond to one kind in extension primer mixture 5~8)；Single base extension condition is：

The detecting step of the kit is：

(1) using the DNA of the sample to be tested of extraction as template, multiplexed PCR amplification reaction is carried out.As described above more Weight PCR reaction systems use amplimer mixture 1 and amplimer mixture 2 to prepare two multiplexed PCR amplification reactions respectively System, multiplexed PCR amplification reaction condition as described above carry out multiplexed PCR amplification reaction, it is anti-to obtain multiplexed PCR amplification Answer product A (corresponding amplimer mixture 1) and B (corresponding amplimer mixture 2).

(2) SAP processing reactions are carried out to multiple PCR products.SAP processing reaction systems as described above prepare 8 parts SAP handles reaction system, wherein multiplexed PCR amplification reaction product A is added in 4 parts, it is anti-that multiplexed PCR amplification is in addition added in 4 parts Answer product B.SAP processing reaction steps as described above carry out SAP processing reactions, obtain SAP and handle reaction product A1, A2, A3, A4 (corresponding multiplex PCR amplified reaction product A) and B1, B2, B3, B4 (corresponding multiplexed PCR amplification reaction product B).

(3) single base amplification is carried out to SAP processing products.Single base extension system as described above, with SAP handles reaction product A1, A2, A3, and A4, B1, B2, B3, B4 8 parts of single base extension systems of preparation, SAP, which is handled, to react Corresponding extension primer mixture 1 and extension termination the mixed liquor G, SAP of being added handles reaction product A2 in the reaction system of product A1 Reaction system in it is corresponding extension primer mixture 2 is added and extends terminate mixed liquor A, SAP handles the reaction of reaction product A3 Corresponding extension primer mixture 3 and the extension termination mixed liquor T, SAP of being added is handled in the reaction system of reaction product A4 in system It corresponds to and extension primer mixture 4 is added and extends corresponding in the reaction system for terminating mixed liquor T, SAP processing reaction product B1 Extension primer mixture 5 is added and extends corresponding be added in the reaction system for terminating mixed liquor C, SAP processing reaction product B2 and prolongs It stretches primer mixture 6 and extends to correspond in the reaction system for terminating mixed liquor G, SAP processing reaction product B3 and extension primer is added It is corresponding in the reaction system of mixture 7 and extension termination mixed liquor G, SAP processing reaction product B4 that extension primer mixture is added 8 terminate mixed liquor A with extension, and single base extension step as described above carries out single base extension.

(4) genotype that each site is directly detected by the method for MALDI-TOF-MS, judges whether to mutate.

Application examples 1

The sample of lung cancer gene mutation feminine gender is detected using kit described in embodiment 1.It is tried used in detection process Agent further includes：The nucleic acid extraction or purification kit of Guangzhou Da Rui Biotechnology Ltd., high pressure sterilization purified water, Absolute ethyl alcohol.

Specific detecting step is as follows：

1. collection of specimens, transport and preservation.

Collection of specimens：Paraffin-embedded tissue sample (FFPE), flesh tissue sample.

It preserves and transports：It is 3 years that paraffin-embedded tissue sample, which answers the dust-proof storage of room temperature and transport, Storage period,；

Flesh tissue sample can in -20 ± 5 DEG C of storages, sample transport using curling stone is on the rocks or bubble chamber it is on the rocks seal into Row transport.

2. detecting step and interpretation of result.

(1) genomic DNA of paraffin-embedded tissue sample and flesh tissue sample is extracted：According to kit specification Standard Operating Procedure carries out.

(2) multiplexed PCR amplification：It is carried out according to the method for embodiment 1.

(3) SAP processing：It is carried out according to the method for embodiment 1.

(4) Single base extension：It is carried out according to the method for embodiment 1.

(5) mass spectrograph and data analysis：It is carried out according to the method for embodiment 1.

As a result：The genotype of SNP site is analyzed according to the signal value that mass spectrograph detects, Fig. 1~34 are the 32 of normal person Bar Single base extension primer interpretation of result collection of illustrative plates.Sample is in 34 sites of this 4 genes of EGFR, KRAS, BRAF and PIK3CA It does not mutate.

Application examples 2

The method of Application Example 1 detects EGFR, KRAS, BRAF, PIK3CA gene point mutation situation.

34 parts are chosen through generation sequence verification difference sample the 34 of EGFR, KRAS, BRAF and PIK3CA this 4 genes The lung cancer FFPE samples that a site mutates extract sample according to the standardization program of the commercial DNA extraction kit of selection DNA carries out multiplexed PCR amplification, SAP processing and single base extension then according to 1 method of embodiment, finally carries out mass spectrum Instrument detection and data analysis.

Testing result：34 parts of samples are respectively 34 site mutations of this 4 genes of EGFR, KRAS, BRAF and PIK3CA The positive, and it is consistent with generation sequencing result, it is specifically shown in the following table 2.

Table 2

This method can realize quickly detection EGFR, KRAS, BRAF, PIK3CA gene point mutation situation.Wherein Figure 35~ 63 be respectively the analysis collection of illustrative plates in the mutational site of 1~P34 of sample P.

Application examples 3

The case where method detection EGFR, KRAS, BRAF, PIK3CA gene point mutation 5% of Application Example 1.

It chooses 4 parts and DNA mixing systems is organized by the other cell strain DNA of the corresponding saltant type of corresponding gene and wild-type human gene It is standby to form, and the sample that mutant proportion is 5%, carry out multiplexed PCR amplification, SAP processing and single base according to 1 method of embodiment Extension finally carries out mass spectrograph detection and data analysis.

Testing result：4 parts of samples are respectively 4 site mutation sun of this 4 genes of EGFR, KRAS, BRAF, PIK3CA Property, specifically it see the table below.

Table 3：

Cell strain title	Sample type	Gene	Exon	Cell strain testing result	This detection method result
						HCC827	Cell strain	EGFR	19	COSM6225	COSM6225
SW620	Cell strain	KRAS	2	COSM520	COSM520
						SK-HEP-1	Cell strain	BRAF	15	COSM476	COSM476
HGC-27	Cell strain	PIK3CA	10	COSM760	COSM760

The result shows that：This method can realize that mutation rate is 5% sample standard deviation and can accurately detect.

Sequence table

<110>Guangzhou Da Rui Biotechnology Ltd.

<120>A kind of kit based on 34 mutational sites of MALDI-TOF-MS detection lung cancer

<160> 48

<170> SIPOSequenceListing 1.0

<210> 1

<211> 33

<212> DNA

<213>People (Homo sapiens)

<400> 1

acgttggatg tctctctgtc atagggactc tgg 33

<210> 2

<211> 28

<212> DNA

<213>People (Homo sapiens)

<400> 2

acgttggatg gtgggcctga ggttcaga 28

<210> 3

<211> 28

<212> DNA

<213>People (Homo sapiens)

<400> 3

acgttggatg accatgcgaa gccacact 28

<210> 4

<211> 27

<212> DNA

<213>People (Homo sapiens)

<400> 4

acgttggatg gcagccgaag ggcatga 27

<210> 5

<211> 31

<212> DNA

<213>People (Homo sapiens)

<400> 5

acgttggatg ctcccaacca agctctcttg a 31

<210> 6

<211> 33

<212> DNA

<213>People (Homo sapiens)

<400> 6

acgttggatg gtgccaggga ccttacctta tac 33

<210> 7

<211> 31

<212> DNA

<213>People (Homo sapiens)

<400> 7

acgttggatg gccaggaacg tactggtgaa a 31

<210> 8

<211> 32

<212> DNA

<213>People (Homo sapiens)

<400> 8

acgttggatg cctccttact ttgcctcctt ct 32

<210> 9

<211> 35

<212> DNA

<213>People (Homo sapiens)

<400> 9

acgttggatg tttcttcatg aagacctcac agtaa 35

<210> 10

<211> 34

<212> DNA

<213>People (Homo sapiens)

<400> 10

acgttggatg tcaattctta ccatccacaa aatg 34

<210> 11

<211> 33

<212> DNA

<213>People (Homo sapiens)

<400> 11

acgttggatg atttttatta taaggcctgc tga 33

<210> 12

<211> 30

<212> DNA

<213>People (Homo sapiens)

<400> 12

acgttggatg cgtccacaaa atgattctga 30

<210> 13

<211> 30

<212> DNA

<213>People (Homo sapiens)

<400> 13

acgttggatg tgagcaagag gctttggagt 30

<210> 14

<211> 31

<212> DNA

<213>People (Homo sapiens)

<400> 14

acgttggatg tgtggaatcc agagtgagct t 31

<210> 15

<211> 35

<212> DNA

<213>People (Homo sapiens)

<400> 15

acgttggatg gctagagaca atgaattaag ggaaa 35

<210> 16

<211> 35

<212> DNA

<213>People (Homo sapiens)

<400> 16

acgttggatg ctccatttta gcacttacct gtgac 35

<210> 17

<211> 15

<212> DNA

<213>People (Homo sapiens)

<400> 17

gtggacaacc cccac 15

<210> 18

<211> 15

<212> DNA

<213>People (Homo sapiens)

<400> 18

aagatcaaag tgctg 15

<210> 19

<211> 17

<212> DNA

<213>People (Homo sapiens)

<400> 19

agcctacgtg atggcca 17

<210> 20

<211> 20

<212> DNA

<213>People (Homo sapiens)

<400> 20

acttgtggta gttggagctg 20

<210> 21

<211> 25

<212> DNA

<213>People (Homo sapiens)

<400> 21

aagttaaaat tcccgtcgct atcaa 25

<210> 22

<211> 27

<212> DNA

<213>People (Homo sapiens)

<400> 22

atttccttgt tggctttcgg agatgtt 27

<210> 23

<211> 15

<212> DNA

<213>People (Homo sapiens)

<400> 23

tgatggccag cgtgg 15

<210> 24

<211> 18

<212> DNA

<213>People (Homo sapiens)

<400> 24

ttctcttccg cacccagc 18

<210> 25

<211> 21

<212> DNA

<213>People (Homo sapiens)

<400> 25

aaattcccgt cgctatcaag g 21

<210> 26

<211> 24

<212> DNA

<213>People (Homo sapiens)

<400> 26

ggatttcctt gttggctttc ggag 24

<210> 27

<211> 15

<212> DNA

<213>People (Homo sapiens)

<400> 27

tgtccacgct ggcca 15

<210> 28

<211> 17

<212> DNA

<213>People (Homo sapiens)

<400> 28

gttggctttc ggagatg 17

<210> 29

<211> 20

<212> DNA

<213>People (Homo sapiens)

<400> 29

ttcccgtcgc tatcaaggaa 20

<210> 30

<211> 17

<212> DNA

<213>People (Homo sapiens)

<400> 30

cgtcgctatc aaggaat 17

<210> 31

<211> 19

<212> DNA

<213>People (Homo sapiens)

<400> 31

tgttgtccag ccaccatga 19

<210> 32

<211> 20

<212> DNA

<213>People (Homo sapiens)

<400> 32

tgattttggt ctagctacag 20

<210> 33

<211> 23

<212> DNA

<213>People (Homo sapiens)

<400> 33

tgtcaagatc acagattttg ggc 23

<210> 34

<211> 25

<212> DNA

<213>People (Homo sapiens)

<400> 34

tttccttgtt ggctttcgga gatgt 25

<210> 35

<211> 15

<212> DNA

<213>People (Homo sapiens)

<400> 35

cctgctcagt gattt 15

<210> 36

<211> 16

<212> DNA

<213>People (Homo sapiens)

<400> 36

gccgaacgca ccggag 16

<210> 37

<211> 18

<212> DNA

<213>People (Homo sapiens)

<400> 37

ccaccgtgca gctcatca 18

<210> 38

<211> 26

<212> DNA

<213>People (Homo sapiens)

<400> 38

ttccttgttg gctttcggag atgttg 26

<210> 39

<211> 17

<212> DNA

<213>People (Homo sapiens)

<400> 39

cggcacacgt gggggtt 17

<210> 40

<211> 19

<212> DNA

<213>People (Homo sapiens)

<400> 40

acttgtggta gttggagct 19

<210> 41

<211> 26

<212> DNA

<213>People (Homo sapiens)

<400> 41

aagttaaaat tcccgtcgct atcaag 26

<210> 42

<211> 27

<212> DNA

<213>People (Homo sapiens)

<400> 42

catcgaggat ttccttgttg gctttcg 27

<210> 43

<211> 15

<212> DNA

<213>People (Homo sapiens)

<400> 43

aggcggcaca cgtgg 15

<210> 44

<211> 18

<212> DNA

<213>People (Homo sapiens)

<400> 44

tggtagttgg agctggtg 18

<210> 45

<211> 20

<212> DNA

<213>People (Homo sapiens)

<400> 45

atcctctctc taaaatcact 20

<210> 46

<211> 24

<212> DNA

<213>People (Homo sapiens)

<400> 46

aggatttcct tgttggcttt cgga 24

<210> 47

<211> 18

<212> DNA

<213>People (Homo sapiens)

<400> 47

gtgatggcca gcgtggac 18

<210> 48

<211> 24

<212> DNA

<213>People (Homo sapiens)

<400> 48

gaggatttcc ttgttggctt tcgg 24

Claims

1. one group of primer combination for detecting 34 mutational sites of lung cancer, which is characterized in that primer is combined by multiplexed PCR amplification React primer and Single base extension primer composition；The sequence such as SEQ ID NO of the multiplexed PCR amplification reaction primer：1~16 institute Show, the sequence such as SEQ ID NO of Single base extension primer：Shown in 17~48.

2. application of the primer combination described in claim 1 in the kit for preparing 34 mutational sites of detection lung cancer.

3. a kind of kit based on 34 mutational sites of MALDI-TOF-MS detection lung cancer, which is characterized in that the kit Including primer combination described in claim 1.

4. kit according to claim 3, which is characterized in that the detection architecture of the kit is by multiplexed PCR amplification Reaction system, SAP handle reaction system and single base extension system composition；Wherein, multiplexed PCR amplification reaction system is by 2 A independent multiplexed PCR amplification reaction system composition, a multiplexed PCR amplification reaction system use amplimer mixture 1, separately One multiplexed PCR amplification reaction system uses amplimer mixture 2；Single base extension system is by 8 independent single alkali Base extension system form, 8 independent single base extension systems respectively use extension primer mixture 1,2,3,4, 5、6、7、8；

SEQ ID NO：1~6, multiplexed PCR amplification reaction primer shown in 11~12 and 15~16 forms amplimer mixture 2；

Amplimer mixture 1 coordinates extension primer mixture 1~4 to use；

Amplimer mixture 2 coordinates extension primer mixture 5~8 to use.

5. kit according to claim 4, which is characterized in that the kit includes multiplexed PCR amplification reaction examination Agent, SAP reagent treatments, Single base extension reagent.

6. kit according to claim 4, which is characterized in that multiplexed PCR amplification reaction system is：6.50 μ L purifying Water, 2.5 μ L multiplex PCR buffer solutions, 2 μ L magnesium chloride buffer solutions, 0.5 μ L dNTP mixtures, 1 μ L multiplex PCR enzymes, 2.5 μ L amplifications 2,10 μ L DNA of primer mixture 1 or amplimer mixture.

7. kit according to claim 4, which is characterized in that the condition of multiplexed PCR amplification reaction is to carry out successively such as Lower step：

（1）95 DEG C 2 minutes；

（2）95 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 1 minute, 45 cycle

（3）72 DEG C 5 minutes；

（4）4 DEG C of preservations.

8. kit according to claim 4, which is characterized in that SAP handles reaction system and is：5 μ L multiplexed PCR amplifications Reaction product, 1.53 μ L purified waters, 0.17 μ L phosphatase buffers, 0.3 μ L phosphoric acid digestion enzymes；It is 37 that SAP, which handles reaction condition, DEG C 40 minutes, 85 DEG C inactivated 5 minutes.

9. kit according to claim 4, which is characterized in that single base extension system is：7 μ L SAP processing are anti- Product, 0.62 μ L high pressure sterilization purified waters, 0.20 μ L is answered to extend buffer solution, 0.04 μ L catalytic reaction enzymes, 0.20 μ L, which extend, to be terminated One kind in mixed liquor A, T, C, G, one kind in 0.94 μ L extension primers mixture 1~8；

Wherein, extend that terminate mixed liquor A, T, C, G be ddATP, ddTTP, ddCTP and ddGTP these four dideoxy nucleotides Mixture aqueous solution, wherein extend terminate mixed liquor A ratio be 1:4:4:4, it is 4 to extend termination mixed liquor T ratios:1:4: 4, the ratio for extending termination mixed liquor C is 5:5:1:5, the ratio for extending termination mixed liquor G is 3:3:3:1；

The reacting hole correspondence of extension primer 1,6 and 7 uses extension to terminate mixed liquor G, the reacting hole pair of extension primer 2 and 8 Extension should be used to terminate mixed liquor A；The reacting hole correspondence of extension primer 3 and 4 uses extension to terminate mixed liquor T, prolongs The reacting hole correspondence of object 5 of extending uses extension to terminate mixed liquor C.

10. kit according to claim 4, which is characterized in that the condition of single base extension is to carry out successively such as Lower step：

（1）Step S1；

（2）Step S2；

（3）Step S3, step S4, step S5 successively, and recycle 5 times；

（4）It carries out successively（2）With（3）Cycle 40 times；

（5）4 DEG C of preservations；

Wherein, step S1：95 DEG C 30 seconds, step S2：95 DEG C 5 seconds, step S3：52 DEG C 5 seconds, step S4：80℃ 5 Second, step S5：72 DEG C 3 seconds.