CN104480215A - Gene joint detection method and kit - Google Patents

Abstract

The invention relates to a joint detection method for gene mutation. The joint detection method is characterized in that 16 gene mutations, including KRAS (Kirsten rat sarcoma viral oncogene homolog), PIK3CA (phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha), BRAF (B-Raf proto-oncogene, serine/threonine kinase) and EGFR (Epidermal Growth Factor Receptor) genes can be detected synchronously by using 20-50ng of DNA as a sample. The method comprises the steps of designing a specific probe; building a reaction system for an MLPA (Multiplex Ligation Dependent Probe Amplification) mutant gene sequences; largely amplifying the targeted gene sequences by PCR (Polymerase Chain Reaction). The joint detection method for gene mutation has the advantages that the specificity is high, and 10-100ng of wild type DNA do not generate nonspecific interference signals; the selectivity is outstanding, and 0.1 to 1.0% of mutation can be detected under the background of 99-99.9% of wild type DNA.

Description

A kind of gene association detection method and test kit

Technical field

The present invention relates to biology field, be specifically related to a kind of method of joint-detection transgenation, particularly one is with MLPA method for technology platform, detects KRAS, PIK3CA, BRAF and EGFR gene 16 kinds of methods of suddenling change altogether simultaneously.

Background technology

RAS gene comprises KRAS, HRAS and NRAS tri-kinds, the mainly KRAS gene relevant to lung cancer, be positioned on No. 12 karyomit(e)s, coding P21 albumen, affect the mcroorganism molecule syntheses such as intracellular nucleic enzyme by acceptance and transfer cell outgrowth stimulus signal, and then the growth and differ entiation of cell is regulated and controled.When No. 2,3, KRAS gene extron is undergone mutation, the KRAS gene of sudden change can sustained activation RAF-MEK-ERK-MAPK signal transduction pathway, causes undesired cell proliferation, differentiation and regulation and control disorderly.In the tumours such as leukemia, lung cancer, large bowel cancer and carcinoma of the pancreas, all found that KRAS gene has higher mutation rate.

Phosphatidyl-inositol 3-kinase (phosphatidylinositol-3-kinase, PI3K) is an important signal transduction pathway molecule, regulates subunit p85 to form by a catalytic subunit p110 and.3 hypotypes such as I type, II type and III type can be divided into according to the p110 constructional feature of PI3K is different with substrate molecule.The signal transduction pathway of PI3K mediation plays an important role in the generation of the propagation of cell, conversion, apoptosis and tumour, the propagation of regulate tumor cell and survival.Research finds, the imbalance of PI3K-AKT signal path can cause the generation of the malignant tumours such as lung cancer, ovarian cancer, breast cancer, glioblastoma, carcinoma of endometrium and nasopharyngeal carcinoma.

BRAF is one of most important proto-oncogene of the mankind, and BRAF sudden change occurs the human tumor of about 8%.BRAF the overwhelming majority sudden change occur in BRAF protein kinase active region or its near, wherein about 70% ~ 90% is that the 1799th Nucleotide T of BRAF gene exons 15 sports A, and the α-amino-isovaleric acid causing it to encode is replaced (namely BRAF V600E suddenlys change) by L-glutamic acid.BRAF gene is positioned at No. 7 karyomit(e)s, it is the serine/threonine kinase in cell mitogen element former activated protein kinase (MAPK) signal path, once this genetic expression changes, biological function will be made to get muddled, thus the signal path making it mediate imbalance also finally cause cell transformation even to be cancerated.

EGF-R ELISA (EGFR) is a kind of tyrosine kinase receptor (RTK), is positioned at No. 7 karyomit(e), is made up of 28 exons, and 1186 amino acid of encoding, its glycoprotein molecule amount is about 170kDa.EGFR is one of I type tyrosine kinase acceptor gene family member, is that transfer cell external signal is to endonuclear important channel albumen.The key signal transduction approach of EGFR has: RAS-RAF-MEK-ERK-MAPK path, PI3K-PDK path, PLC-γ path, JAK-STAT path etc.By these approach, extracellular signal is converted into intracellular signal, thus successfully manages extraneous token stimulus, regulate the growth of cell, propagation, differentiation, the apoptosis of T suppression cell.On front four exons that the sudden change of EGFR mainly occurs in TK region in born of the same parents (18 ~ 21), the TK region mutagenesis found at present has kind more than 30.Classical sudden change or hot spot mutation are again in alternative sudden change (L858R) wherein on the deletion mutantion (delE746-A750) of exons 19 and exon 21, account for 90% of sudden change.In addition the alternative mutation T 790M occurred on extron 20 is medicament-resistant mutation, in Molecular Detection, have important reference value equally.

Multiplex ligation-dependent probe amplification (multiplex ligation-dependent probeamplification, MLPA) is a kind of Multiple detection technology of carrying out quantitative and semi-quantitative analysis for DNA sequence dna to be checked first reported by Schouten etc.In MLPA reaction, each target sequence that increases needs a pair probe, and each probe comprises one section of primer sequence and one section of specific sequence.After this pair probe and target sequence are hybridized, then be connected to become an oligonucleotide chain with ligase enzyme.Ligation high special, only have when two probes and target sequence are hybridized completely, i.e. target sequence and probe-specific sequence complete complementary, two sections of probes could be connected into a complete single nucleic acid strands by ligase enzyme; Otherwise, if target sequence and probe sequence are not exclusively complementary, even if only have the difference of a base, also can cause hybridization not exclusively, ligation cannot be carried out.After ligation completes, to increase the probe connected with a pair universal primer, the length of the amplified production of each probe is unique, and scope is between 130 ~ 480bp.MLPA technology can be used for Multiple detection, by rational probe design, can detect multiple target sequence in primary first-order equation simultaneously.When adopt capillary electrophoresis amplified production is separated, analyze, the target sequence simultaneously detected can reach 45.Multiple detection method is conducive to improving detection efficiency, reduces the usage quantity of template, is particularly useful for the few and situation that target spot to be measured is more of template amount.If the target sequence origination point sudden change detected or disappearance, amplification sudden change, so the amplified peak of correspondent probe just can lack, reduces or increase, therefore, just can judge whether target sequence has the exception of copy number or point mutation existence according to the change of amplified peak.

Before formulation clinical treatment, application molecular diagnostic techniques detects each transgenation state in patient's tumour cell exactly, thus select suitable target therapeutic agent, be that application targeted drug carries out prerequisite and the basis of personalized treatment to cancer, the prognosis that simultaneously also can be clinical judge patient is offered help.The special targeted drug for EGFR, PIK3CA, BRAF enters Clinical practice at present, and for the targeted drug of KRAS also in research and development.In lung cancer patient, KRAS, PIK3CA, BRAF and the modal mutant form of EGFR gene have 16 kinds, if use the method for quantitative fluorescent PCR to detect, then need a large amount of clinical samples, not only add the difficulty of clinical sampling, also make patient pay higher testing cost.In order to reduce the usage quantity of clinical sample, simplify and detect operation, the invention provides a kind of based on MLPA technology, efficiently, highly sensitive detection method, KRAS, PIK3CA, BRAF and EGFR gene totally 16 kinds of sudden changes can be detected, for clinical individual medication provides foundation in the DNA sample of 20-50ng simultaneously.

Summary of the invention

In order to improve Clinical Laboratory ability, now provide a kind of based on MLPA technology, the methods that can detect the 16 kinds of sudden changes altogether of KRAS, PIK3CA, BRAF and EGFR gene simultaneously, the method can detect 16 kinds of sudden changes in the DNA sample of 20-50ng simultaneously.

Technical scheme of the present invention comprises following steps:

1. detect gene mutation site

According to wildtype gene sequence and the corresponding mutated genes sequence of No. 2, mankind KRAS gene, 3 exons, design multipair specific probe, detect the catastrophe in following site:

According to wildtype gene sequence and the corresponding mutated genes sequence of No. 9, mankind PIK3CA gene, 20 exons, design multipair specific probe, detect the catastrophe in following site:

According to wildtype gene sequence and the corresponding mutated genes sequence of mankind's BRAF gene 15 exon, design specific probe, detect BRAF V600E sudden change.Namely the 1799th Nucleotide T of 15 exons sports A, and the α-amino-isovaleric acid causing it to encode is replaced (Cosmic ID 476) by L-glutamic acid.

According to wildtype gene sequence and the corresponding mutated genes sequence of No. 19, human EGFR gene, No. 20 and 21 exons, design specific probe, detect alternative sudden change L858R (the Cosmic ID 6224 on the deletion mutantion of 19 exons, 21 exons, alternative mutation T 790M on c.2573T>G) and 20 exons (Cosmic ID 6240, c.2369C>T).

2. probe design process

Need a pair probe to detect target sequence, contain with the complementary district of target sequence strictly complementary in the structure of probe, regulate the fill area of probe length, the guiding region as PCR primer.The primer sequence in PCR primer district can be universal primer or Auele Specific Primer.Can not comprise sudden change in probe or comprise sudden change, mutating alkali yl position within the probe can be positioned at last (when complementary district is positioned at 3 ' end of probe) or first (when complementary district is positioned at 5 ' end of probe).A pair probe is after ligase enzyme connects, and length is between 100 ~ 250nt.

3.19 exon deletion mutantion detection methods

EGFR gene 19 exon easily occur lack region c.2230_2270 designing probe to A, when region c.2230_2270 occur disappearance time cannot amplify strips A; On EGFR gene conserved sequence, designing probe is to B.When not comprising the disappearance in EGFR 19 exon c.2230_2270 region in sample DNA, a certain proportion of A, B two kinds of bands can be amplified.When disappearance containing EGFR 19 exon c.2230_2270 region in sample DNA, the ratio of A band and B band changes, thus judges that EGFR gene 19 exon there occurs deletion mutantion.

4. set up the reaction system of MLPA amplification mutant gene sequence, react a large amount of amplifying target genes sequence by PCR.MLPA reaction system is made up of following reaction:

Template denaturation: added by template DNA in PCR pipe, is placed in PCR instrument, and set temperature is 95 ~ 100 DEG C, maintains 3 ~ 10min, then is cooled to 25 DEG C.

Hybridization: the mixture adding MLPA Buffer and probe in template after denaturing.The concentration of its middle probe is 0.5 ~ 10nM; Containing Tris-HCl (50 ~ 500mM, pH8.2 ~ 9.0) in MLPA Buffer, KCl (2 ~ 5M), MgCl2 (0.5 ~ 5M), EDTA (0.5 ~ 5mM).PCR instrument runs hybridization procedures: 95 DEG C of 1min; 60 DEG C of 16 ~ 24h.

Connect: after hybridization terminates, in each PCR pipe, add ultrapure water and ligase enzyme, PCR instrument runs linker: 50 ~ 58 DEG C of 10 ~ 30min; 95 ~ 100 DEG C of 10 ~ 30min; Be cooled to 20 DEG C.

Pcr amplification: ligation terminates the amplification of laggard performing PCR.In each PCR pipe, add PCR universal primer, dNTPs and Taq enzyme, PCR instrument run PCR program:

Detect: the methods such as agarose electrophoresis, polyacrylamide gel electrophoresis and capillary electrophoresis can be adopted to detect the nucleic acid fragment of amplification.

The invention provides a kind of for detecting the test kits detecting the 16 kinds of sudden changes altogether of KRAS, PIK3CA, BRAF and EGFR gene simultaneously, the specific probe in test kit is in table 1.

Table 1: specific probe sequence table

Described do not comprise sample DNA extraction step based on MLPA technology, the methods that detect the 16 kinds of sudden changes altogether of KRAS, PIK3CA, BRAF and EGFR gene simultaneously, but for fixing the DNA fragmentation that paraffin-embedded sample obtains from formaldehyde, still there is the amplification same with the DNA extracted from fresh tissue sample and detectivity.

Compared with existing detection method, the present invention has following advantage:

(1) detect KRAS, PIK3CA, BRAF and EGFR gene 16 kinds of sudden changes altogether simultaneously;

(2) high specificity, the wild-type DNA of 10 ~ 100ng can not produce nonspecific interference signal;

(3) selectivity is strong, can detect the saltant type of 0.1% ~ 1.0% under the wild-type DNA background of 99 ~ 99.9%;

(4) amount of samples is low, with 20-50ng DNA for sample can detect KRAS, PIK3CA, BRAF and EGFR gene 16 kinds of sudden changes altogether simultaneously.

Accompanying drawing explanation

Fig. 1: embodiment 1 polyacrylamide gel electrophoresis figure

Fig. 2: embodiment 2 polyacrylamide gel electrophoresis figure

Fig. 3: embodiment 3 polyacrylamide gel electrophoresis figure

Embodiment

With specific embodiment, the present invention is described in further detail below.Should be understood that, specific embodiment is only make clearer explanation to the present invention, instead of limitation of the present invention.

Embodiment 1

Respectively get DNA 2 μ L in table 2 as template in PCR pipe, be placed in PCR instrument, set temperature is 95 DEG C, maintains 5min, then is cooled to 25 DEG C.Add 1.5 μ L MLPABuffer and 1.5 μ L Probemix (containing 34 probes in table 1 in Probemix) in template after denaturing, PCR instrument runs hybridization procedures: 95 DEG C of 1min; 60 DEG C of 16h.After hybridization terminates, in each PCR pipe, add 15 μ L ultrapure waters and 0.1 μ L ligase enzyme, PCR instrument runs linker: 54 DEG C of 20min; 95 DEG C of 10min; Be cooled to 20 DEG C.Ligation terminates the amplification of laggard performing PCR.In each PCR pipe, add 1.0 μ LPCR universal primers, 1.0 μ L dNTPs and 0.5 μ L Taq enzyme, PCR instrument run PCR program:

Detected the nucleic acid fragment of amplification by polyacrylamide gel electrophoresis, see Fig. 1.Each catastrophe point is all accurately detected, and the nucleotide fragments length of pcr amplification is consistent with the length of probe.

Table 2: embodiment 1 template DNA

Embodiment 2

Respectively get DNA 2 μ L in table 3 as template in PCR pipe, all the other checked operations, with embodiment 1, are detected the nucleic acid fragment of amplification, see Fig. 2 by polyacrylamide gel electrophoresis.When the DNA extracted in healthy human blood is as sample, pcr amplification goes out two band that length is 186bp and 194bp; And with E746-A750 disappearance HCC827 cell genomic dna for sample time, only amplify the band that length is 194bp.

Table 3: embodiment 2 template DNA

Embodiment 3

Respectively get DNA 2 μ L in table 4 as template in PCR pipe, all the other checked operations, with embodiment 1, are detected the nucleic acid fragment of amplification, see Fig. 3 by polyacrylamide gel electrophoresis.When there is several genes sudden change in sample simultaneously, this method of inspection can out by the gene test of sudden change simultaneously.

Table 4: embodiment 3 template DNA

Embodiment 4

Collect the tumor tissues (comprising flesh tissue and paraffin section) that clinical diagnosis is 57 routine patients of lung cancer, use the DNA extraction kit of Qiagen company and paraffin embedding DNA extraction kit to extract genomic dna as template.All the other checked operations, with embodiment 1, detect the nucleic acid fragment of amplification by capillary electrophoresis.The transgenation situation detected in 57 routine samples is in table 5.

Table 5: Tumor Tissues of Patients with Lung Cancer DNA detection in Gene Mutation result

In addition the sample in embodiment 4 is carried out sequence verification, the detected result of sequencing result and the present embodiment is completely the same.

Claims (7)

1. a gene association detection method, can detect KRAS, PIK3CA, BRAF and EGFR gene 16 kinds of sudden changes altogether simultaneously.

2. gene association detection method as claimed in claim 1, it is characterized in that, the sudden change of detection comprises:

KRAS gene mutation:

PIK3CA transgenation:

BRAF gene mutation: V600E (Cosmic ID 476)

EGFR genetic mutation: the deletion mutantion of 19 exons, L858R (Cosmic ID 6224, c.2573T>G) and T790M (Cosmic ID 6240, c.2369C>T).

3. gene association detection method as claimed in claim 1, is characterized in that, contain table 1middle P-SEQ-1 ~ P-SEQ-34 specific probe.

4. a gene association detection kit, is characterized in that, comprises specific probe described in claim 3.

5. a kind of gene association detection kit as claimed in claim 4, comprises following operation steps:

Template denaturation: added by template DNA in PCR pipe, is placed in PCR instrument, and set temperature is 95 ~ 100 DEG C, maintains 3 ~ 10min, then is cooled to 25 DEG C;

Hybridization: the mixture adding MLPA Buffer and probe in template after denaturing, the concentration of its middle probe is 0.5 ~ 10nM; Containing Tris-HCl (50 ~ 500mM, pH8.2 ~ 9.0) in MLPA Buffer, KCl (2 ~ 5M), MgCl2 (0.5 ~ 5M), EDTA (0.5 ~ 5mM); PCR instrument runs hybridization procedures: 95 DEG C of 1min; 60 DEG C of 16 ~ 24h;

Connect: after hybridization terminates, in each PCR pipe, add ultrapure water and ligase enzyme, PCR instrument runs linker: 50 ~ 58 DEG C of 10 ~ 30min; 95 ~ 100 DEG C of 10 ~ 30min; Be cooled to 20 DEG C;

Pcr amplification: ligation terminates the amplification of laggard performing PCR, adds PCR universal primer, dNTPs and Taq enzyme, PCR instrument is run PCR program in each PCR pipe;

Detect: the methods such as agarose electrophoresis, polyacrylamide gel electrophoresis and capillary electrophoresis can be adopted to detect the nucleic acid fragment of amplification.

6. a kind of gene association detection kit as claimed in claim 4, detected sample comprises fresh pathological tissue, paraffin embedding pathological tissue, paraffin section, saliva, sputum, urine and blood.

7. for detecting probes for KRAS, PIK3CA, BRAF and EGFR gene 16 kinds of sudden changes altogether, it is characterized in that, being made up of the oligonucleotide of at least 70% identity the oligonucleotide probe of P-SEQ-1 ~ P-SEQ-34 or sequence and its.