WO2011131145A1

WO2011131145A1 - Liquid chip for detecting egfr gene mutations

Info

Publication number: WO2011131145A1
Application number: PCT/CN2011/073210
Authority: WO
Inventors: 许嘉森; 李国强; 余刚; 秦会娟
Original assignee: 广州益善生物技术有限公司
Priority date: 2010-04-23
Filing date: 2011-04-23
Publication date: 2011-10-27
Also published as: CN102234683B; CN102234683A

Abstract

The invention belongs to biochip technical field and discloses a liquid chip, for detecting EGFR gene mutations, comprising: (i) allele-specific primer extension (ASPE) primer pair(s) designed for both wild-type and mutant EGFR gene, where each ASPE primer comprises a tag sequence at 5' end and a specific primer part at 3' end, which is aiming at the mutant site of target gene; (ii) microspheres coated separately with a specific anti-tag sequence and encoded with different colors, where the said anti-tag sequence matches correspondingly with the said tag sequence; and (iii) amplification primers for amplifying target sequence(s) with mutant site(s) of Exon 19, Exon 20 and /or Exon 21. The detection method provided by the present invention has a coincidence rate up to 100% with sequencing methods. The liquid chip for detecting EGFR gene mutations prepared is excellent in signal to noise ratio, and cross reaction between designed probes and anti-tag sequences is basically free.

Description

EGFR gene mutation detection liquid phase chip

The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to a liquid phase chip for detecting mutation of EGFR gene. technical background

Epidermal growth factor receptor (EGFR) is a multifunctional glycoprotein widely distributed on the cell membrane of human tissues. It has tyrosine kinase activity and is one of the four members of the Ε family. Named HER1 or ErbBl. When EGFR is activated by a ligand, it initiates intracellular signaling, and through a cascade of adaptor proteins and enzymes in the cytoplasm, regulates the transcription of transcription factor-activated genes, directing cell migration, adhesion, proliferation, differentiation, and apoptosis. Studies have shown that high expression or abnormal expression of EGFR exists in many solid tumors, which provides a theoretical basis and experimental basis for EGFR-targeted tumor therapy and signal transduction intervention for EGFR signaling pathway.

Currently, there are two main types of molecularly targeted drugs developed for EGFR: 1) Small molecule tyrosine kinase inhibitors (TKI), such as Gefitinib and Erlotinib, inhibit EGFR cells. Internal region tyrosine kinase activity; 2) Monoclonal antibodies (MAb), such as Cetuximab and Panitumumab, bind to the extracellular domain of EGFR and block ligand-dependent EGFR activation. These drugs block EGFR-mediated intracellular signaling pathways through different pathways, thereby inhibiting tumor growth, metastasis and angiogenesis, and promoting tumor cell apoptosis and increasing chemosensitivity.

Studies have shown that the efficacy of gefitinib and other molecularly targeted drugs targeting EGFR is significantly associated with mutations in the EGFR gene. More than 90% of the EGFR gene mutations found so far are located in exons 19-21. These mutations can be divided into 3 types:

1) Exon 19 base deletion

The exon 19 mutation is mainly a base deletion mutation at codons 746 to 752, resulting in the loss of the amino acid (ELREATS) sequence in the EGFR protein, which changes the receptor ATP-binding pocket (ABP). Angle, which significantly enhances the sensitivity of cancer cells to gefitinib. Studies have shown that the exon 19 gene mutation has the highest incidence rate, accounting for more than 50% of the total number of EGFR gene mutations. According to incomplete statistics, there are at least 22 different types of exon 19 gene mutations that have been discovered at home and abroad.

The exon 19 mutation mainly has the following deletion types, and the specific sequence thereof is as described in the table, wherein, relative to the wild type, the sequence of the mutant deletion is represented by a "-" sign:

Genotype deletion type sequence (5'-3')

Wild type Wild-type GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAAT TAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGAT

Ml del E746-A750 GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAA

( 1 ) --- AACATCTCCGAAAGCCAACAAGGAAATCCTCGAT

M2 del E746-A750 GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAG

(2) —— ACATCTCCGAAAGCCAACAAGGAAATCCTCGAT

M3 del GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAA—

L747-E749ins P --- C-CAACATCTCCGAAAGCCAACAAGGAAATCCTCGAT

M4 del GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAA—

L747-A750ins P C-CATCTCCGAAAGCCAACAAGGAAATCCTCGAT

M5 del L747-T751 GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAA—

TCTCCGAAAGCCAACAAGGAAATCCTCGAT

M6 del GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGG

L747-T751ms A CATCTCCGAAAGCCAACAAGGAAATCCTCGAT

M7 del L747-S752 GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAA—

CATCTCCGAAAGCCAACAAGGAAATCCTCGAT

M8 del GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGG-TT--

L747-S752ins V CCGAAAGCCAACAAGGAAATCCTCGAT

(2)

M9 del GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGA---

L747-S752ins D TCCGAAAGCCAACAAGGAAATCCTCGAT

M10 del GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAA—

L747-S752ms Q CA-GAAAGCCAACAAGGAAATCCTCGAT

(2)

Mi l del GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAAT-

L747-S752ins S CGAAAGCCAACAAGGAAATCCTCGAT

M12 delE749-S752ins GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAAT

D (GAT) TAAGAGA TCCGAAAGCCAACAAGGAAATCCTCGAT

M13 delE746-T751in GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAG

s T (ACC) —― -ACC-TCTCCGAAAGCCAACAAGGAAATCCTCGAT M14 delE746-P753ins GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGG VS (GTC TCG) TCT-CGAAAGCCAACAAGGAAATCCTCGAT

M15 delE746-T751in TATCCCAGAAGGTGAGAAAGATAAAATTCCCGTCGCTATCAAGG-TAT CA s Y (TAT) TCTCCGAAAGCCAACAAGAAT

M16 GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAA—

CA-ATCTCCGAAAGCCAACAAGGAAATCCTCGAT

M17 delE746-T751in GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGG-CT-- s A (GCT) TCTCCGAAAGCCAACAAGGAAATCCTCGAT

M18 delE746-A750in ATCCCAGAAGGTGAGAAAGATAAAATTCCCGTCGCTATCAAGG-CCC CAACA s AP(GCC TCTCCGAAAGCCAACAAGGAA

CCA)

M19 del GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGG

L747-T751ins V T-ATCTCCGAAAGCCAACAAGGAAATCCTCGAT

2) Mutation type of exon 20

The mutation in exon 20 is mainly due to the C-T conversion at codon 790, which causes the threonine at this position in the EGFR protein to be replaced by methionine (T790M). This mutation was only seen in patients who relapsed after drug treatment, and the mutation caused cancer cells to develop resistance to gefitinib and erlotinib. In addition, mutations that occur in exon 20 are also base insertions, usually occurring at codons 770-775, and at 0 (( between 0 010 00 00 sequences (mainly on both sides of the CCCCC sequence)) Different insertion methods, the inserted fragments are 3 to 9 bases, but the practical application significance of such insertion mutations is still unclear.

3) Point mutation of exon 21

The point mutation in exon 21 is mainly due to the T-G conversion at codon 858, which causes the leucine at this site in the EGFR protein to be converted to arginine (L858R). This mutation is located near the DFG sequence. Its role is to increase the stability of A-loop, and the sensitivity of cancer cells to gefitinib and erlotinib is significantly enhanced.

At present, the established EGFR gene mutation detection technology is mainly PCR-sequencing and real-time fluorescent quantitative PCR detection technology. PCR-sequencing has the advantage of being able to determine the range and type of mutations. It is currently widely used, but the sensitivity of sequencing is only 20%-25%, which is far from meeting the needs of practical applications, especially for heterogeneity. Mutations in tumor somatic cells, low sensitivity will lead to a large number of missed tests. At the same time, the sequencing detection process is complicated and the timeliness is poor. For the practical application detection requiring high timeliness and high sensitivity, the limitations of the sequencing method have been highlighted. Real-time fluorescent quantitative PCR The detection technology has high detection efficiency and strong timeliness, but its false positive rate is also a problem for practical applications. Based on the problems of the above detection techniques, the "Detection probe for EGFR gene mutation sites, liquid phase chip and its detection method" successfully developed by the applicant in the early stage (Application No.: 200810027613.9) The detection method is simple and convenient, and is excluded by enzymatic digestion. The interference caused by a large number of wild-type sequences has good specificity, high sensitivity and accuracy of more than 99%. However, this method involves two rounds of PCR operation, which is easy to cause pollution, and the hybridization detection step probes are close to each other (only the mutation position Different points), it is not convenient to detect multiple gene mutation sites in parallel. Summary of the invention

One of the objects of the present invention is to provide a EGFR gene mutation detecting liquid phase chip. The liquid phase chip can be used to detect the normal genotype of exon 19 of EGFR gene and its 19 major deletion mutants: M1~M19, normal genotype of exon 20 and mutant T790M, and normal of exon 21. Genotype and mutant L858R.

The technical solution to achieve the above objectives is as follows:

A liquid phase chip for detecting EGFR gene mutations, mainly comprising:

(A) Wild-type and mutant ASPE bow pairs designed for mutation sites of the EGFR gene: Each ASPE primer consists of a 5'-end tag sequence and a 3'-end specific primer for the target gene mutation site. The tag sequence is selected from the sequences of SEQ ID NO. 1 to SEQ ID NO. 24; the specific primer is: a specific primer for the Exon 19 mutant, the specific primer is derived from SEQ ID NO. 26~ one or more base sequences of SEQ ID NO. 44, or one of SEQ ID NO 74-SEQ ID NO. 92 (SEQ ID NO: 26 - SEQ ID NO: 4) The above base sequence, the 3' end of each specific primer is derived from 1 to 6 bases adjacent to the 3' end of the corresponding deletion region, and can specifically recognize the corresponding mutation type, and for the wild type of Exon 19 Specific primer, the specific primer is derived from SEQ ID

a base sequence in N0.25 or its reverse complement sequence, and its 3' end contains a unique deletion base in the deletion region of each mutant to be detected, and is not related to the specific primer of all mutants. The same; and/or for Exon 20, derived from the base sequence in SEQ ID NO. 45 to SEQ ID NO. 46 or its reverse complement; and/or against Exon 21, derived from SEQ ID N0.47- a base sequence in SEQ ID N0.48 or its reverse complement; one of the last 3 bases of the 3' end of the specific primer for Exon 20 and Exon 21 is a mutation site or its corresponding wild Type site; the Tm value of all the above specific primers is between 52 58 ;;

(B) microspheres having different color coding, respectively, coated with a specific anti-tag sequence, the anti-tag sequence being selected from the sequences of SEQ ID N0.121 to SEQ ID N0.144, and the anti The -tag sequence can be complementarily paired with the tag sequence selected in (A);

(C) Amplification primers for amplifying a target sequence having a corresponding mutation site of Exon 19, Exon 20 and/or Exon 21, respectively. Preferably, the amplification primer is: SEQ ID NO. 145 146 for Exon 19; and for Exon 20, when the specific primer is derived from SEQ ID NO. 45 to SEQ ID NO. 46, the amplification primer is SEQ ID NO. When the specific primer is derived from the reverse complement of SEQ ID N 0.45 to SEQ ID NCX46, the amplification primer is SEQ ID NO. 149-150; for Exon 21, when the specific primer is derived from SEQ ID NO. 47 to SEQ ID NO. At 48 o'clock, the amplification primers are SEQ ID N0.151-152, and when the specific primers are derived from the reverse complement of SEQ IDN0.47~SEQIDN0.48, the amplification primer is SEQ ID NO. 153 154.

Preferably, the specific primer for Exon 19 is: one or more of SEQ ID NO. 50-68, or more than one of SEQ ID N 0.98 116, and SEQ ID NO: 0.97 „ for Exon 20 The specific primers are: SEQ ID NO. 69-70, or SEQ ID 0.117-118. Specific primers for Exon 21 are: SEQ ID N0.71~72, or SEQ

Another object of the present invention is to provide a specific primer for the detection of mutations in the EGFR gene.

The technical solution to achieve the above objectives is as follows:

Specific primers for detecting EGFR gene mutations are: specific primers for the Exon 19 mutant, which are derived from more than one base sequence in SEQ IDN 0.26 to SEQ IDN 0.44, respectively, or derived from the reverse To a base sequence of one or more of the complementary sequences SEQ IDN0.74 to SEQ ID N0.92, the 3' end of each specific primer is derived from 1 to 6 bases adjacent to the 3' end of the corresponding deletion region, And specifically recognize the corresponding mutation type, and a specific primer for Exon 19 wild type, the specific primer is derived from the base sequence in SEQ ID N0.25 or its reverse complement, and its 3, end contains a specific deletion base in the deletion region of each mutant to be detected, and which is different from the specific primers of all mutants; or against Exon 20, derived from SEQ IDN0.45 to SEQ ID N0.46 or a base sequence in its reverse complement sequence; and/or a base sequence derived from Exon 21, derived from SEQ ID NO. 47 to SEQ ID NO. 48 or its reverse complement; specific primers for Exon 20 and Exon 21 One of the last 3 bases of the 3' end Group is a mutation site or its corresponding wild-type site; all of the above specific I bow Tm value was between 52 ~ 58 ° C.

The main advantages of the invention are:

1. The coincidence rate between the detection method and the sequencing method provided by the invention is as high as 100%. The prepared EGFR gene mutation detection liquid phase chip has a very good signal-to-noise ratio, and there is substantially no cross-reaction between the designed probe and the anti-tag sequence.

2. The specific primers designed by the present invention have very good specificity and can accurately distinguish various mutation types. Various specific ASPE primers are capable of performing hybridization reactions under uniform reaction conditions, and there is no substantial existence between various primers and probes. Non-specific binding.

3. The detection method of the invention has simple steps, and the amplification of the target sequence of multiple sites can be completed by one-step multiplex PCR, thereby avoiding many uncertain factors existing in complicated operations such as repeated PCR, and thus can greatly Improve the detection accuracy, reflecting the precise simultaneous qualitative and quantitative analysis features.

4. The detection result of the invention is stable and reliable, and the combination of various ASPE-specific primers enables the liquid phase chip and the detection method to form a system with good detection effect. detailed description

Example 1 EGFR gene mutation detection liquid phase chip, mainly includes:

First, ASPE primers

The normal genotype of exon 19 of EGFR gene and its 19 major deletion mutants: M1 M19, the normal genotype of exon 20 and the mutant T790M, and the normal genotype of exon 21 and the mutant L858R, Specific primer sequences were designed separately.

The design points of the ASPE bow of the EGFR gene mutation detection are:

The ASPE primer consists of a "Tag + specific primer sequence". Among them, the 5' end is a Tag sequence designed based on EGFR gene mutation detection. The designed Tag sequence can avoid the secondary structure that ASPE primers may form in the reaction system, and the Tag sequence and Tag sequence, Tag sequence and specificity. There is no cross-reactivity between the sex primer sequences. The Tag sequence and the specific primer sequence form a complete ASPE, and all the ASPEs can be synchronized in a uniform reaction system (ie, the buffering environment of the same reaction, the same reaction temperature, etc.), and the parallel detection is completed. . The designed Tag sequence is as shown in Table 1.

The 3' end is a specific primer sequence designed according to the mutation detection of EGFR gene, and the specific primer has a Tm value of 52 to 58 ° C; specific primers for the Exon 19 mutant are derived from SEQ ID N0, respectively. .26 in the base sequence of the 44 or its reverse complement, the 3' end of each specific primer is derived from 1 to 6 bases adjacent to the 3' end of the deletion type of the mutation type (according to the set Tm The value is between 52 and 58 °C, and the approximate number of bases is determined, that is, the specific primer covers the bases at both ends of the deletion region), and the corresponding mutation type can be specifically identified; the specificity for the Exon 19 wild type Primer, the specific primer is derived from the base sequence SEQ ID N0.73 in SEQ ID N0.25 or its reverse complement, and the 3' end contains all (each) of the deleted regions of the mutant to be detected. The unique deletion base, for example, the wild type specific primer in Table 3, SEQ ID 0.49, covers the unique bases of the 19 mutations to be detected, and the wild type and various mutant specific primers are different from each other. , can specifically extend the sequence of various types. Specific primer sequences for various mutants of Exon 20 or Exon 21, one of the last three bases at the 3' end is a mutation site; the specific primer sequence of the Exon 20 or Exon 21 wild type, One of the last 3 bases of the 3' end is the corresponding wild type site of the mutation site. The Tm value is calculated as Tm = (G+C) 4+ ( Α + Τ ) χ 2 - 4. Tag sequence I

Detection of specific sequence of EGFR gene mutation (Εχ ₀ η19 is a deletion mutation, Εχ _Ο η20, 21 is a point mutation, wherein, within the mutation site)

SEQ ID Type The sequence of the specific primer (5'_3')

NO.

GTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCT

25 19-w (wild type)

CCGAAAGCCAACAAGGAAATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAAACATCTCCGAAAGCCAACAAG

26 19M1 (mutant type)

GAAATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGACATCTCCGAAAGCCAACAAG

27 19M2 (mutant type)

GAAATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGGAACCAACATCTCCGAAAGCC

28 19M3 (mutant type)

AACAAGGAAATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGGAACCATCTCCGAAAGCCAAC

29 19M4 (mutant type)

AAGGAAATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGGAATCTCCGAAAGCCAACAAG

30 19M5 (mutant type)

GAAATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGGCATCTCCGAAAGCCAACAAG

31 19M6 (mutant type)

GAAATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGGAACATCTCCGAAAGCCAACA

32 19M7 (mutant type)

AGGAAATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGGTTCCGAAAGCCAACAAGGAA

33 19M8 (mutant type)

ATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGGATCCGAAAGCCAACAAGGAA

34 19M9 (mutant type)

ATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGGAACAGAAAGCCAACAAGGA

35 19M10 (mutant type)

AATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGGAATCGAAAGCCAACAAGGAA

36 19M11 (mutant type)

ATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGATCCGAAAGCC

37 19M12C mutant type)

AACAAGGAAATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGACCTCTCCGAAAGCCAACAAG

38 19M13 C mutant)

GAAATCCTCGAT GTTAAAATTCCCGTCGCTATCAAGGTCTCGAAAGCCAACAAGGAA

39 19M14C mutant)

ATCCTCGAT

GATAAAATTCCCGTCGCTATCAAGGTATCATCTCCGAAAGCCAAC

40 19M15 C mutant)

AAGAAT

GTTAAAATTCCCGTCGCTATCAAGGAACAATCTCCGAAAGCCAAC

41 19M16 mutant)

AAGGAAATCCTCGAT

GTTAAAATTCCCGTCGCTATCAAGGCTTCTCCGAAAGCCAACAAG

42 19M17 (mutant type)

GAAATCCTCGAT

GATAAAATTCCCGTCGCTATCAAGGCCCCAACATCTCCGAAAGCC

43 19M18C mutant type)

AACAAGGAA

GTTAAAATTCCCGTCGCTATCAAGGTATCTCCGAAAGCCAACAAG

44 19M19 mutant)

GAAATCCTCGAT

45 20-w (wild type) CTCCACCGTGCAGCTCATCA@GCAGCTCATGCCCTTCGGCT

T790M (mutation

46 CTCCACCGTGCAGCTCATCA|GCAGCTCATGCCCTTCGGCT

Type)

47 21-w (wild type) GTCAAGATCACAGATTTTGGGC|GGCCAAACTGCTGGGTGCG

48 L858R (mutant) GTCAAGATCACAGATTTTGGGC back GGCCAAACTGCTGGGTGCG According to the design points of the specific primer sequences in the above ASPE primers, a series of specific primer sequences were designed, and some wild type and mutant specific primers were exemplified, as shown in Table 3:

Table 3 EGFR gene mutation detection specific primer sequence one

SEQ Tm value exon type specific primer sequence (5'-3')

ID NO.

Exon CTATCAAGGAA773⁄4^G^G^GC 56

49 19 19-w or TATCAAGGAA7 3⁄4viG^G^GG4 54

ATCAAGGAA7 GA GAAGCA 54

AATTCCCGTCGCTATCA C 56

50 19M1 or ATTCCCGTCGCTATCAA^4G4 56

TTCCCGTCGCTATCAA/i4 C4r 56 TTCCCGTCGCTATCAAG CA 56

51 19M2 or TCCCGTCGCTATCAAGL G4r 56 Person CCCGTCGCTATCAAG/4 47 54

CGTCGCTATCAAGGAACG4yi 56

52 19M3 or GTCGCTATC AAGGAACGL4 C 56 Person TCGCTATCAAGGAACG4^C4 54

CGTCGCTATCAAGGAACG4 T 56

53 19M4 or GTCGCTATCAAGGAACG4rC 56 TCGCTATCAAGGAAC 4rCr 54

CGTCGCTATCAAGGAArCr 56

54 19M5 or CGTCGCTATCAAGGAArCrC 56 GTCGCTATCAAGGAArCrCC 56

CCCGTCGCTATCAAGGC47 56

55 19M6 or CCGTCGCTATCAAGGG47U 56 persons CGTCGCTATCAAGGC47Tr 54

CCGTCGCTATCAAGGAAC4 54

56 19M7 or CGTCGCTATCAAGGAAG47T 56 persons CCCGTCGCTATCAAGGAAC 56

CCCGTCGCTATCAAGGTTC 56

57 19M8 or CCGTCGCTATCAAGGTTCC 56 CGTCGCTATCAAGGTTCCG 56

CCGTCGCTATCAAGGArCC 56

58 19M9 or CCCGTCGCTATCAAGGA7U 56 GTCGCTATCAAGGA7UC& 54

CGTCGCTATCAAGGAAG4 G 54

59 19M10 or CCGTCGCTATCAAGGAAC4 54 By CCCGTCGCTATCAAGGAAC 56

60 19M11 CGTCGCTATCAAGGAATCG 54 Or GTCGCTATCAAGGAATCG 4 56 by CCGTCGCTATCAAGGAATC 54

CTATCAAGGAATTAAGAGA7CC 56

19M12 or GCTATCAAGGAATTAAGAGArC 56 TATCAAGGAATTAAGAGArCCG 56

TCCCGTCGCTATCAACL4 CC 56

19M13 or CCCGTCGCTATCAA04CC 56 person CCGTCGCTATC AAG CCTC 56

CCCGTCGCTATCAAGGrCr 56

19M14 or CCCGTCGCTATCAAGGrCrC 56 CGTCGCTATCAAGGrCrCG 56

CCGTCGCTATCAAGGTATC4 56

19M15 or CGTCGCTATCAAGGTATC 7 54 by GTCGCTATC AAGGTATC4 TC 54

CCGTCGCTATCAAGGAAC L4 56

19M16 or CGTCGCTATCAAGGAAGH 54 GTCGCTATCAAGGAAC4^rC 54

CCCGTCGCTATCAAGGCTT 56

19M17 or CCGTCGCTATCAAGGCT7C 56 persons CGTCGCTATCAAGGCT7Tr 54

TCGCTATCAAGGCCCG4.4C 56

19M18 or GTCGCTATCAAGGCCCGW 56 CGTCGCTATCAAGGCCCC 56

CGTCGCTATCAAGG73⁄47Tr 52

19M19 or CGTCCATACCAAGG3⁄47U7U 56 person CCCGTCGCTATCAAGGE47 ¹ 54

Exon 20-w CACCGTGCAGCTCATCA@ 54 20 or CCGTGCAGCTCATCA@GC 56 ACCGTGCAGCTCATCA^ 54

CCACCGTGCAGCTCATCA| 56

70 T790M or CCGTGCAGCTCATCA GC 56

ACCGTGCAGCTCATCA|G 54

CAAGATCACAGATTTTGGGCT 56

71 21-w or GATCACAGATTTTGGGC0GG 56

Exon AAGATCACAGATTTTGGGC|G 56

21 AAGATCACAGATTTTGGGC^ 54

72 L858R or ATCACAGATTTTGGGC^GG 54

AGATCACAGATTTTGGG(3⁄4G 56

As shown in the table, the specific primer sequence of the Exonl9 deletion mutation is detected, and the 3' end of each specific primer is derived from 1 to 6 bases adjacent to the 3' end of the deletion type of the mutation type, and the base is in italics. Indicates that the point at which ΕΧΟΠ20 and Exon21 are mutated is marked with 闺.

Similarly, the reverse complement of the sequence of the specific primers in Table 2 can also be used to design specific primers for ASPE. The specific sequence options are shown in Table 4:

EGFR gene mutation detection source specific sequence of I

SEQ

Explicit

ID type The sequence of the specific primer (5 '-3')

Child

NO.

Exon 19-w ATCGAGGATTTCCTTGTTGGCTTTCGGAGATGTTGCTTCTC

73

19 (wild type) TTAATTCCTTGATAGCGACGGGAATTTTAAC

19M1 ATCGAGGATTTCCTTGTTGGCTTTCGGAGATGTTTTGATAG

74

(mutant type) CGACGGGAATTTTAAC

19M2 ATCGAGGATTTCCTTGTTGGCTTTCGGAGATGTCTTGATAG

75

(mutant type) CGACGGGAATTTTAAC

19M3 ATCGAGGATTTCCTTGTTGGCTTTCGGAGATGTTGGTTCCT

76

(mutant type) TGATAGCGACGGGAATTTTAAC

19M4 ATCGAGGATTTCCTTGTTGGCTTTCGGAGATGGTTCCTTGA

77

(mutant type) TAGCGACGGGAATTTTAAC 19M5 ATCGAGGATTTCCTTGTTGGCTTTCGGAGATTCCTTGATAG

(mutant type) CGACGGGAATTTTAAC

19M6 ATCGAGGATTTCCTTGTTGGCTTTCGGAGATGCCTTGATAG

(mutant type) CGACGGGAATTTTAAC

19M7 ATCGAGGATTTCCTTGTTGGCTTTCGGAGATGTTCCTTGAT

(mutant type) AGCGACGGGAATTTTAAC

19M8 ATCGAGGATTTCCTTGTTGGCTTTCGGAACCTTGATAGCGA

(mutant type) CGGGAATTTTAAC

19M9 ATCGAGGATTTCCTTGTTGGCTTTCGGATCCTTGATAGCGA

(mutant type) CGGGAATTTTAAC

19M10 ATCGAGGATTTCCTTGTTGGCTTTCTGTTCCTTGATAGCGA

(mutant type) CGGGAATTTTAAC

19M11 ATCGAGGATTTCCTTGTTGGCTTTCGATTCCTTGATAGCGA

(mutant type) CGGGAATTTTAAC

19M12 ATCGAGGATTTCCTTGTTGGCTTTCGGATCTCTTAATTCCT

(mutant type) TGATAGCGACGGGAAT1TTAAC

19M13 ATCGAGGATTTCCTTGTTGGCTTTCGGAGAGGTCTTGATA

(mutant type) GCGACGGGAATTTTAAC

19M14 ATCGAGGATTTCCTTGTTGGCTTTCGAGACCTTGATAGCG

(mutant type) ACGGGAATTTTAAC

19M15 ATTCTTGTTGGCTTTCGGAGATGATACCTTGATAGCGACG

(mutant type) GGAATTTTATC

19M16 ATCGAGGATTTCCTTGTTGGCTTTCGGAGATTGTTCCTTGA

(mutant type) TAGCGACGGGAATTTTAAC

19M17 ATCGAGGATTTCCTTGTTGGCTTTCGGAGAAGCCTTGATA

(mutant type) GCGACGGGAATTTTAAC

19M18 TTCCTTGTTGGCTTTCGGAGATGTTGGGGCCTTGATAGCG

(mutant type) ACGGGAATTTTATC

19M19 ATCGAGGATTTCCTTGTTGGCTTTCGGAGATACCTTGATA (mutant type) GCGACGGGAATTTTAAC

AGCCGAAGGGCATGAGCTGC§TGATGAGCTGCACGGTGG

93 20-w (wild type)

Exon AG

20 AGCCGAAGGGCATGAGCTGC^TGATGAGCTGCACGGTGG

94 T790M (mutant)

AG

95 Exon 21-w (wild type) CGCACCCAGCAGTTTGGCC prisoner GCCCAAAATCTGTGATCTTGAC

96 21 L858R (mutant) CGCACCCAGCAGTTTGGCC^GCCCAAAATCTGTGATCTTGAC

Similarly, according to the design points of the specific primer sequences in the ASPE primers described above, a series of specific primer sequences were designed from the source sequences of the specific primer sequences in Table 4, among which some wild-type and mutant-specific primers were exemplified. , as shown in Table 5:

EGFR gene mutation detection specific primer sequence two

SEQ

Exon type specific primer sequence (5'-3') Tm ( °C ) ID NO.

Exon TCGGAGATGTTGC7TC7T773⁄4 56

97 19 19-w (wild type) or CGGAGKTGTJGCJTCTCTTAA 56

GGAGATGTJGCITCTCTTAATT 56

TTGGCTTTCGGAGATGTT7TG 56

98 19M1 (mutant) or GGCTTTCGGAGATGTT7TGL r 56

TGGCTTTCGGAGATGTT7TG4 56

TTGGCTTTCGGAGATGTC7T 54

99 19M2 (mutant) or TGGCTTTCGGAGATGTC7TG 56

TGTTGGCTTTCGGAGATGTC 56

GCTTTCGGAGATGTTGG7TC 56

100 19M3 (mutant) or TTTCGGAGATGTTGG7TCC 54

CTTTCGGAGATGTTGG7TCC 56

101 19M4 (mutant) TGTTGGCTTTCGGAGATGG7 56 Or GTTGGCTTTCGGAGATGG7T 56 by TTGGCTTTCGGAGATGG7TC 56

TTGTTGGCTTTCGGAGArrC 54

19M5 (mutant type) or TGTTGGCTTTCGGAGA7TCC 56 person GTTGGCTTTCGGAGA7TCC 56

TTGTTGGCTTTCGGAGATGC 56

19M6 (mutant type) or GTTGGCTTTCGGAGATGCC 56 person TTGGCTTTCGGAGATGCCrr 56

GTTGGCTTTCGGAGATG7TC 56

19M7 (mutant type) or TTGTTGGCTTTCGGAGATG7T 56 person TTGGCTTTCGGAGATG7TCC 56

TCCTTGTTGGCTTTCGG^ C 56

19M8 (mutant type) or CTTGTTGGCTTTCGG CC 54 person ATTTCCTTGTTGGCTTTCGOL4 56

CCTTGTTGGCTTTCGGA7T 56

19M9 (mutant type) or CTTGTTGGCTTTCGGArCC 56 person TTGTTGGCTTTCGGA7UC 54

GATTTCCTTGTTGGCTTTCrG 56

19M10 (mutant type) or ATTTCCTTGTTGGCTTTCrG 54 person TTTCCTTGTTGGCTTTC7U7T 54

ATTTCCTTGTTGGCTTTCG47 54

19M11 (mutant type) or TTTCCTTGTTGGCTTTCG^7T 54 person TCCTTGTTGGCTTTCG^ rrc 54

CTTGTTGGCTTTCGGArcrC 56

19M12 (mutant type) or TTGTTGGCTTTCGGA7U7U7T 56 person TTGTTGGCTTTCGGATCrCr 54

19M13 (mutant) TTGTTGGCTTTCGGAGAGG 56 or GTTGGCTTTCGGAGAGGTC 56 TTGGCTTTCGGAGAGGrCr 54

CTTGTTGGCTTTCGAGACC 54

19M14 (mutant type) or TTGTTGGCTTTCGAGACC7 54 person TGTTGGCTTTCGAGACC7TG 56

GTTGGCTTTCGGAGATG4E4 54

19M15 (mutant type) or TTGGCTTTCGGAGATCL4E4C 54 person TGGCTTTCGGAGAT04 CC 56

TTGTTGGCTTTCGGAGATrG 54

19M16 (mutant type) or TGTTGGCTTTCGGAGAT rGr 54 person GTTGGCTTTCGGAGAT7G7T 54

TTGTTGGCTTTCGGAGAvl GC 56

19M17 (mutant type) or GTTGGCTTTCGGAGA^ GCC 56 person TTGGCTTTCGGAGA^ GCCTT 56

TTGGCTTTCGGAGATGTTGG 56

19M18 (mutant type) or GGCTTTCGGAGATGTTGGG 56 person GCTTTCGGAGATGTTGGGG 56

TGTTGGCTTTCGGAGAL4CC 56

19M19 (mutant type) or GTTGGCTTTCGGAGAL4 CCT 56 person TGGCTTTCGGAGAL4 CCTTG 56

CGAAGGGCATGAGCTGC^ 56

20-w (wild type) or AAGGGCATGAGCTGC back to TG 54

Exon GAAGGGCATGAGCTGC^T 54 20 CGAAGGGCATGAGCTGCQ 54

T790M (mutant type) or AAGGGCATGAGCTGC^TG 54 person GAAGGGCATGAGCTGC T 54

Exon CACCCAGCAGTTTGGCC^ 54 21 21-w (wild type) or CCCAGCAGTTTGGCC prison GC 56 person ACCCAGCAGTTTGGCC prisoner G 54 CACCCAGCAGTTTGGCC 56

120 L858R (mutant) or CCAGCAGTTTGGCC^GC 54

ACCCAGCAGTTTGGCC^G 56 All ASPE primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. Each of the synthesized primers was formulated into a storage solution of 100 pmol/mL with 10 mmol/L Tris Buffer.

Second, the anti-tag sequence coated microspheres

According to the designed ASPE-specific primer sequence, the tag sequence was selected to minimize the secondary structure of the anti-tag sequences of each microsphere and the possible formation of the tag and ASPE-specific primer sequences. The selected 24 microsphere numbers were selected. The corresponding anti-tag sequences on the microspheres are shown in Table 6.

Table 6 Microsphere number and corresponding anti-tag sequence on the microsphere

SEQ ID

Corresponding anti-tag sequence on the microsphere (5'-3') microsphere number

NO.

121 ATTATGAAGTAAGTTAATGAGAAG 47

122 AAAGGATTAAAGTGAAGTAATTGA 33

123 AAAGGTAAGATTATTGATGAAAAG 65

124 ATTATTGAGATGTGAAGTTTGTTT 48

125 ATTGTTGATGATTGATTGAAATGA 51

126 ATTGTGAAGTATAAAGATGATTGA 52

127 TGAAAAAGTGTAGATTTTGAGTAA 26

128 ATGAATTGAAAGTGATTGAAAAAG 54

129 GTAATGATAAAGATGATGATATTG 57

130 ATGAGATTATTGGATTTGTAGATT 60

131 ATTGTTGAATTGATGAGATTTGAT 73

132 ATGATGATGTATTGTAGTTATGAA 79

133 TGATGTAAGTATTGATGTTAGTTT 87

134 TGATTTGAGTATTTGAGATTTTGA 18 135 GTAGTAATGTTAATGAATTAGTAG 58

136 TGATTGAATTGAGTAAAAAGGATT 22

137 TTGATAATGTTTGTTTGTTTGTAG 28

138 GTAGATAGTATAGTTGTAATGTTA 66

139 AAAGGTAAGATTATTGATGAAAAG 55

140 ATTGAAAGATGAAAAGATGAAAAG 37

141 GTATTGTATTGAAAAGGTAATTGA 24

142 GTATAAAGAAAGATTGGTAAATGA 44

143 TGTAGAAGATGAGATGTATAATTA 53

144 TGAAGATTTGAAGTAATTGAAAAG 25

The 24 microspheres selected were purchased from Luminex, USA, and the anti-tag sequence was coated on the microspheres. The anti-tag sequence and the microspheres are connected with a 5-10 T-spacer sequence, that is, a 5-10 T-spacer sequence is added before each anti-tag sequence, and the anti-tag sequence is generated by Shanghai. Engineering Bioengineering Technology Services Co., Ltd. Synthesis. The synthetic anti-tag sequences with sterile ddH ₂ 0 stock solution was formulated 100nmol / ml of. The spacer arm is a sequence for spacing the anti-tag from the surface of the microsphere or placing the anti-tag in a hydrophilic environment. By setting an appropriate length of the spacer sequence between the anti-tag sequence and the microspheres, the steric hindrance can be reduced, the efficiency of the hybridization reaction, and the specificity of the hybridization reaction can be improved. Common spacer sequences include poly dT, poly (dT), oligotetraethylene glycol, and (CH2) n spacer arms (n ≥ 3), such as (CH2) 12, (CH2) 18 and the like. In addition, if poly (dA) interference is present, poly (TTG) can also be used as the spacer arm. The spacer arm of the present invention preferably has 5-10 T.

The process of microsphere coating is as follows:

5 x 10 ⁶ carboxylated microspheres of the above number (purchased from Luminex) were suspended in 50 ul of 0.1 mol/L MES solution (pH 4.5), and 10 μl of synthetic anti-tag molecule (100 nmol/ml) was added. ). A 10 ng/ml EDC (N-(3-dimethylaminopropyl)-N-ethylcarbodiimide) (purchased from Pierce Chemical) working solution was prepared. Add 2.5 ul of EDC working solution to the microsphere suspension, incubate for 30 minutes at a constant temperature, add 2.5 ul of EDC working solution, and incubate for 30 minutes at constant temperature. After the reaction was completed, it was washed once with 0.02% Tween-20 and once with 0.1% SDS solution. The washed microspheres coated with the anti-tag sequence were resuspended in 100 μl of Tris-EDTA solution [10 mmol/L Tris (pH 8.0), 1 mmol/L EDTA], and stored at 2-8 ° C in the dark. 3. Amplifying a primer having a target sequence of a detection site

The normal genotype of the EGFR gene Exonl9 and its 19 major deletion mutants were detected: M1~M19, the normal genotype of Exon20 and the mutant T790M, and the normal genotype of Exon21 and the mutant L858R. Three pairs of primers (see Table 7) were designed using Primer 5.0 to amplify the target sequence with the detection site.

Exon20 and Exon21 mutation sites were detected, and when the specific primer sequence was derived from SEQ.N045 48, the sequence containing the target site was amplified using SEQ ID NO. 147-148 and SEQ ID N0.151-152; When the primer sequence is derived from SEQ ID N0.93-96, the sequence containing the target site is amplified using SEQ ID NO. 149-150 and SEQ ID N 0.153-154. Detection of Exonl9 mutation sites was performed using SEQ ID N 0.145-146 to amplify the sequence containing the target site. The designed amplification primer pair can complete the multiplex PCR reaction simultaneously under the same PCR amplification condition, which greatly improves the detection efficiency.

Table 7 Primers for amplifying the target sequence of the EGFR gene mutation site

All primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. Each primer after synthesis is used separately

10 mmol/L Tris Buffer was formulated into a 100 pmol/mL stock solution.

Example 2 Detection of samples by liquid crystal chip using EGFR gene mutation detection

The formulations of the various solutions are as follows: 50 mM MES Buffer (pH 5.0) Formulation (250 ml):

After filtration, it was stored at 4 °C.

The ExoSAP-IT kit was purchased from the US USB company.

Biotin-labeled dCTP was purchased from Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

First, the sample DNA extraction:

Refer to "Molecular Cloning" for DNA extraction related methods to obtain DNA to be detected.

2. PCR amplification of the sample to be tested

Primer 5.0 was designed with primers, and multiplex PCR was performed in two tubes. Three target sequences containing the detection sites were amplified in one step. The amplification primers for Exonl9, Exon20 and Exon21 were SEQ ID NO. 145-146, respectively. SEQ ID NO. 147-148 and SEQ ID NO. 151-152, the product sizes are 117 bp, 141 bp, 131 bp, respectively _; when the amplification primers of Exonl9, Exon20 and Exon21 are SEQ ID N0.145-146, SEQ, respectively When ID No. 149-150 and DSEQ ID N0.153-154, the product sizes were 117 bp, 168 bp, and 156 bp, respectively. The primer sequences are shown in Table 7 above.

First, prepare the multiplex PCR primer working solution: Take the primer storage solution of SEQ ID NO. 145-154, respectively, lOOul in a 1.5 ml microcentrifuge tube, and mix well to obtain the multiplex PCR primer working solution. The PCR reaction system is as follows:

Ιθχ buffer (containing Mg ²⁺ ) 5 ul

dNTP (2.5mmol/L each) 4 ul

Taq enzyme (5U/ul) 0.5 ul

Multiplex PCR primer working solution (16.17pmol/mL each) 6 ul

Template DNA ( lOng/ul) 1 ul

ddH ₂ 0 33.5 ul

50 ul The PCR amplification procedure was: 95 °C for 3 min _; 94 °C for 20 s, 56 °C for 30 s, 72 °C for 30 s, 30 cycles; 72 °C for 10 min; 4 °C for storage.

Third, the enzyme product digestion treatment

The detailed steps are as follows:

1. Take 7.5 ul of PCR product and add 3 ul of ExoSAP-IT enzyme;

2. Incubate for 15 min at 37 °C. Incubate at 80 ° C for 15 min to inactivate excess enzyme. The digested product was used directly in the subsequent ASPE primer extension reaction.

Site-specific primer extension reaction (ASPE)

The primer extension reaction was carried out using the ASPE-specific primer designed above, and biotin-labeled dCTP was incorporated during the reaction, so that the product after the reaction was labeled with multiple biotin labels.

First, prepare the mixed ASPE primer working solution: Take the corresponding wild type and mutant ASPE primers of Exonl9, Exon20 and Exon21 (specifically shown in Table 8), and store the lOul in 2 different 1.5ml microcentrifuge tubes and add 10mmol. /L Tris Buffer is added to 200ul, and evenly mixed is the ASPE mixed primer working solution. The ASPE reaction system is as follows:

For the detection of Exonl9, Exon20 and Exon21 mutants, Groupl, Group3 and Group 5 perform ASPE primer extension reaction in the same tube, and the extension template is a PCR product containing the target site, and the amplification primers of the PCR product are respectively SEQ ID N0.145-146, SEQ ID N0.147-148 and SEQ ID NO. 151-152; likewise, Group2, Group 4 and Group6 perform ASPE primer extension reaction in the same tube, and the extension template contains the target site. The PCR product, the amplification primers for this PCR product are SEQ ID NO. 145-146. SEQ ID NO. 149-150 and SEQ ID N 0.153-154, respectively.

Table 8 Design of EGFR gene mutation detection liquid-phase chip preparation

Genotype experimental group genotype specific primer tag sequence anti-tag sequence ll

Exon9 Group 1 19-w-l SEQ ID N0.49 SEQ ID NO. l SEQ ID NO.

19M1-1 SEQ ID NO. 50 SEQ ID NO. 2 SEQ ID NO. 122

19M2-1 SEQ ID N0.51 SEQ ID NO.3 SEQ ID NO.123

19M3-1 SEQ ID N0.52 SEQ ID NO. 4 SEQ ID NO. 124

19M4-1 SEQ ID N0.53 SEQ ID NO. 5 SEQ ID NO.

19M5-1 SEQ ID N0.54 SEQ ID NO. 6 SEQ ID NO.

19M6-1 SEQ ID N0.55 SEQ ID NO. 7 SEQ ID NO.

19M7-1 SEQ ID N0.56 SEQ ID NO. 8 SEQ ID NO.

19M8-1 SEQ ID N0.57 SEQ ID NO. 9 SEQ ID NO. 129

19M18-2 SEQ ID NO. 115 SEQ ID NO. 19 SEQ ID NO. 139

19M19-2 SEQ ID NO. 116 SEQ ID NO. 20 SEQ ID NO.

20-w-l SEQ ID N0.69 SEQ ID N0.21 SEQ ID NO. 141

Group3

T790M-1 SEQ ID NO. 70 SEQ ID NO. 2 SEQ ID NO. 142

Exon20

SEQ ID NO.117 SEQ ID NO. 2 SEQ ID NO.

Group4

T790M-2 SEQ ID NO. 118 SEQ ID N0.22 SEQ ID NO. 142

21-w-l SEQ ID N0.71 SEQ ID N0.23 SEQ ID NO. 143

Group5

L858R t-1 SEQ ID N0.72 SEQ ID N0.24 SEQ ID NO.144

Exon21

21-W-2 SEQ ID NO.119 SEQ ID NO. 2 SEQ ID NO.

Group6

L858R-2 SEQ ID NO. 120 SEQ ID NO. 2 SEQ ID NO.

10x buffer 2 ul

MgCl ₂ ( 50mmol/L) 0.5 ul

Biotin-dCTP (400umol/L) 0.25 ul dATP, dGTP, dTTP mixture (each lOOumol/L) 1 ul

Tsp enzyme ( ⁵ U/ul) 0.25 ul mixed ASPE primer working solution (500 nmol/L each) 1 ul Digested PCR amplification product 5 ul ddH ₂ 0 —10. ul Total 20 ul Reaction procedure: 96° C 2min; 94 °C 30s, 52 °C lmin, 72 °C 2min, 30 cycles; 4 °C for storage. Five, hybridization reaction

1. According to the designed ASPE primers, select the corresponding optimal 24 kinds of microspheres (the concentration of microspheres is 2.5x l0 ⁵ / ml). Each microsphere has a different color code;

2. Take lul each numbered microspheres in a 1.5ml microcentrifuge tube;

3. The microspheres are centrifuged at ≥ 10000g for l-2min;

4. Discard the supernatant and resuspend the microspheres in lOOul of 2xTm hybridization buffer and vortex to mix;

5. Take 25 ul of the above microsphere suspension in the corresponding well of a 96-well filter plate, and add 25 ul of ddH ₂ 0 to the control well;

6. Take 5-25 ul of ASPE reaction solution in the corresponding wells, make up to 50 ul with ddH ₂ 0;

7. Wrap the 96-well plate in foil to protect it from light, and incubate at 95 ° C for 60 s, 37 ° C for 15 min; 8. The microspheres after hybridization are ≥300 (3⁄4 centrifugation for 2-4 min;

9. Remove the supernatant and resuspend the microspheres in 75 ul of l xTm hybridization buffer;

10. The microspheres are centrifuged at ≥3000g for 2_5min;

11 Resuspend the microspheres in 75 ul of l xTm hybridization buffer and add 15 ul of streptavidin-phycoerythrin (SA-PE) at a concentration of 10 ug/ml;

12. Incubate at 37 ° C for 15 min and test on a Luminex instrument.

Sixth, results detection and data analysis

The reacted product was detected by a Luminex series of analytical instruments. The fluorescence value (MFI) is greater than 100 as the cut-off value. When the MFI value of the mutant detection is greater than 100, the sample is determined to have the mutation type, otherwise the sample is determined to be the corresponding wild type, and the detection results are shown in Table 9, Table 10. Table 11 and Table 12 are shown.

The EGFR gene mutation of a large number of samples was detected by the method, and the coincidence rate of the detection results of the method provided by the present invention was calculated by the sequencing method and the liquid phase chip result. The method detects that the EGFR genotype detection results of 20 samples and the sequencing results are 100%. It can be seen that the EGFR gene mutation detection liquid chip provided by the invention can accurately detect the mutation type of the EGFR gene, and the result is stable and reliable. The detection of the samples by the two sets of specific primer sequences designed from the same mutation site and derived from SEQ ID 0.25-48 and its reverse complement sequence SEQ ID N0.73-96, respectively, achieved consistent detection results. Different liquid phase chips designed according to the above-mentioned points of ASPE primer design, for example, other specific sequences exemplified in Table 3 and Table 5, have corresponding specific primer sequences different, and the detection results are consistent. Specific test data for other similar situations is omitted.

Table 9 Sample Test Results 1 (MFI)

Table 10 Sample Test Results 2 (MFI)

Table 11 Sample Test Results 3 (MFI)

Exon20 Exon21

Sample

Group3 Group4 Group5 Group6

T790M-1 T790M-2 21-w-l L858R-1 21-w-2 L858R-2 negative

33 40 38 33 32 39 43 33 Control

1 4315 58 4365 t 62 2275 61 2694 59

2 4213 57 4319 58 2329 62 2398 57

3 4119 59 4417 65 2810 62 2494 56

4 4297 58 4286 51 2857 56 2988 52

5 4408 54 4020 52 2401 60 2877 61

6 4101 60 4097 53 2784 64 2914 55

7 4477 62 4402 54 2433 63 2221 54

8 4343 60 4257 62 2090 52 2632 58

9 4285 52 4227 54 2188 53 2467 57

10 4033 57 4398 60 2009 64 2300 61

11 4012 51 4123 58 2557 63 2043 52

12 4129 52 4087 65 2585 54 2420 54

13 4134 663 4409 654 2563 51 2621 53

14 4267 58 4495 62 2584 60 2232 51

15 4464 53 4273 55 2631 51 2474 59

16 4356 63 4090 61 2559 56 2500 58

17 4089 52 4280 50 2574 1250 2104 1262

18 4047 57 4486 57 2753 61 2899 52

19 4016 57 4135 60 2469 54 2046 60

20 4141 57 4131 57 2836 62 2750 59 Table 12 Analysis of sample EGFR gene mutation detection results

Example 3 Detection of EGFR Gene Mutations by Liquid Chips of Different ASPE Primers

First, the design of liquid phase chip preparation (the selection of Tag sequence and Anti-Tag sequence)

Taking the detection of the 19M1 mutation site in exon 19 of EGFR gene as an example, the specific primer sequence of the 3' end of ASPE primer was designed for wild type (19-w) and 19M1 mutant of exon 19, while ASPE Tag at the 5' end of the primer The sequence is selected from the group consisting of two of SEQ ID NO. l - SEQ ID N0.24, correspondingly, the anti-tag sequence coated on the microsphere complementary to the corresponding tag sequence selects SEQ ID N0.121 - SEQ ID The specific design of NO.144 _{0 is} shown in the following table (Table 13). The synthesis of ASPE primers, anti-tag sequence-coated microspheres, amplification primers, detection methods, and the like are as described in Example 1 and Example 2.

Table 13 Design of liquid phase chip preparation

Second, sample testing

Using the liquid phase chip prepared by the above design, the samples 21-40 were tested according to the detection process and method described in Example 2, and the test results were as follows:

Table 14 Sample test results (MFI) and gene mutation analysis

No. Group 1 Group2 Group3

NO. wild type mutant wild type mutant wild type mutant negative control 31 26 35 26 26 25

21 2815 58 2024 64 1637 57

22 1782 64 2221 62 2895 63

23 2862 1260 2014 1262 2169 1263

24 1732 56 1817 52 2057 51

25 2637 54 1997 50 2100 53

26 2751 53 2883 56 1992 57

27 1910 55 1854 63 2741 59

28 2253 54 2102 56 2293 52

29 2893 1563 1713 1555 1607 1557

30 2633 64 2415 60 2054 51

31 1892 54 1675 57 2790 64

32 1790 54 2359 50 2405 50 33 1822 56 2916 51 2739 53

34 2683 61 2296 61 2533 54

35 2233 63 2357 53 2065 61

36 2673 65 2270 52 1742 55

37 2237 56 2062 62 3016 57

38 2402 51 2940 57 2046 51

39 2260 53 3071 52 2206 58

40 2351 63 2598 64 1921 58 Table 15 Samples Analysis of EGFR gene mutation detection results Liquid chip test results

Sample number sequencing test results

Group 1 Group2 Group3

21 wild type wild type wild type wild type

22 wild type wild type wild type wild type

23 19M1 mutation 19M1 mutation 19M1 mutation 19M1 mutation

24 wild type wild type wild type wild type

25 wild type wild type wild type wild type

26 wild type wild type wild type wild type

27 wild type wild type wild type wild type

28 wild type wild type wild type wild type

29 19M1 mutation 19M1 mutation 19M1 mutation 19M1 mutation

30 wild type wild type wild type wild type

31 wild type wild type wild type wild type

32 wild type wild type wild type wild type

33 wild type wild type wild type wild type

34 wild type wild type wild type wild type

35 wild type wild type wild type wild type

36 wild type wild type wild type wild type

37 wild type wild type wild type wild type

38 wild type wild type wild type wild type 39 wild type wild type wild type wild type

40 Wild type Wild type Wild type Wild type Other liquid phase chips for different mutation sites, ASPE primers using the different tag sequences designed above, the results are still stable and reliable, the specific data is omitted.

Example 4 Selection of wild type and mutant specific primer sequences for exon 19 deletion mutant gene detection

I. Design of liquid phase chip preparation (selection of wild-type and mutant-specific primer sequences)

According to the design point of the EGFR gene Exonl9 deletion mutation-specific primer, the 3' end of the specific primer sequence is derived from the 1 to 6 base adjacent to the 3' end of the deletion mutation region and can specifically recognize the corresponding mutation type. Specific primer sequences for 19-w, 19M1 and 19M2 are shown in Table 16.

Taking the detection of the EGFR gene Exonl9 deletion mutation as an example, the specific primer sequences of the wild type and mutant ASPE primers were designed for the wild type of Εχ ₀ η19 and the 19M1 and 19M2 mutants, respectively. The specific design is shown in the following table (Table 17). The synthesis of ASPE primers, anti-tag sequence-coated microspheres, amplification primers, detection methods, and the like are as described in Example 1 and Example 2.

EGF Exonl9 gene deletion mutation detection specific primer

Table 17 Design of liquid chip preparation III genotype experimental group genotype specific primer tag sequence anti-tag sequence ll

19-w-l SEQ ID NO 49 SEQ ID NO. 1 SEQ ID NO.

Grou 1 19M1-1 SEQ ID NO. 50 SEQ ID NO. 2 SEQ ID NO. 122

EGFR 19M2-1 SEQ ID N0.51 SEQ ID NO. 3 SEQ ID NO.

(Exonl9) SEQ ID NO. 155 SEQ ID NO. 1 SEQ ID NO.

Group2 19M1-2 SEQ ID NO. 156 SEQ ID NO. 2 SEQ ID NO. 122

19M2-2 SEQ ID NO. 157 SEQ ID NO. 3 SEQ ID NO. Second, sample testing

Using the liquid phase chip prepared by the above design, the samples 41-60 were tested according to the detection process and method described in Example 2, and the test results were as follows:

Table 18 Sample test results (MFI) and gene mutation analysis

Table 19 Analysis of sample EGFR gene mutation detection results

Liquid chip test results

Sample number sequencing test results

Group 1 Group2

41 wild type wild type wild type

42 wild type wild type wild type 43 wild type wild type wild type

44 wild type wild type wild type

45 19M1 mutation 19M1 mutation 19M1 mutation

46 wild type wild type wild type

47 wild type wild type wild type

48 wild type wild type wild type

49 wild type wild type wild type

50 wild type wild type wild type

51 wild type wild type wild type

52 wild type wild type wild type

53 wild type wild type wild type

54 wild type wild type wild type

55 19M2 mutation 19M2 mutation 19M2 mutation

56 wild type wild type wild type

57 wild type wild type wild type

58 wild type wild type wild type

59 wild type wild type wild type

60 wild-type wild type wild type designed for Exonl9 identical deletion mutation of EGFR, two sets of specific primer sequences derived from SEQ ID N049-51 and SEQ ID N0.155-157, respectively, were tested for consistent detection. effect. According to the above-mentioned ASPE primer design, the 3' end of the specific primer sequence is derived from 1 to 6 bases adjacent to the 3' end of the deletion mutation region, and different liquid crystal chips are designed respectively, and the specific primer sequences are different. The detection results are consistent, but when the 3' end of the specific primer sequence is derived from the 1 to 3 base adjacent to the 3' end of the deletion mutation region (Groupl), the signal-to-noise ratio of the detection result is better, and its specificity And the sensitivity is correspondingly higher. Specific test data for other similar situations is omitted.

The above is a detailed description of the possible embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and the equivalents of the invention are included in the scope of the invention.

Claims

Rights request

1. A liquid phase chip for detecting EGFR gene mutation, characterized in that it mainly comprises:

(A) Wild-type and mutant ASPE bows paired for the EGFR gene mutation sites: The specificity of each ASPE bow from the 5'-end tag sequence and the 3' end against the target gene mutation site The primer sequence is selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 24; the specific primer is: a specific primer for the Exon 19 mutant, the specific primer is derived from SEQ ID NO. More than one base sequence in 26~SEQIDNO.44, or more than one base sequence in the reverse complement sequence, the 3' end of each specific primer is derived from the 3' end of the corresponding deletion region 1 to 6 bases in proximity, and specifically recognize the corresponding mutation type, and a specific primer for Exon 19 wild type, which is derived from the base sequence in SEQ IDN 0.25 or its reverse complement And its 3' end contains a unique deletion base in the deletion region of each mutant to be detected, and is different from the specific primers of all mutants; and/or for Exon 20, derived from SEQ ID N0. 45-SEQ ID N0.46 or its reverse complement Base sequence in ; and/or against Exon 21, base sequence derived from SEQ ID N0.47-SEQ ID N0.48 or its reverse complement; specific primers for Exon 20 and Exon 21 One of the last 3 bases of the 3' end is a mutation site or its corresponding wild type site; the Tm value of all the above specific primers is between 52 and 58 ;;

(Β) respectively coated with different color-coded microspheres having a specific anti-tag sequence, the anti-tag sequence being selected from the sequences of SEQ IDN 0.121 to SEQ IDN 0.144, and the anti-tag sequence Correspondingly paired with the tag sequence selected in (A);

(C) Amplification primers for amplifying a target sequence having a corresponding mutation site of Exon 19, Exon 20 and/or Exon 21, respectively.

2. The EGFR gene mutation detecting liquid phase chip according to claim 1, wherein the amplification primer is: a needle

SEQIDN0.145~146 of X-inch Exon 19; for Exon 20, when the specific primer is derived from SEQ ID N0.45~SEQ ID N0.46, the amplification primer is SEQ IDN 0.147-148, when the specific primer is derived SEQ ID N0.45 The reverse complement of SEQ ID N0.46, the amplification primer is SEQ ID NO. 149^150; for Exon 21, when the specific primer is derived from SEQ ID NO. 47 to SEQ ID NO. 48, amplification The primer is SEQ ID NO. 151 152. When the specific primer is derived from the reverse complement of SEQ ID N0.47 to SEQ IDN 0.48, the amplification primers are SEQ ID Nos. 0.153 to 154.

The EGFR gene mutation detecting liquid phase chip according to claim 1 or 2, wherein the specific primer for the Exon 19 mutant type, the 3' end of each specific primer is derived from the corresponding deletion region 3 '1 to 3 bases adjacent to the end.

The EGFR gene mutation detection liquid phase chip according to claim 1 or 2, characterized in that it is specific to Exon 19. The heterologous primers are: one or more of SEQ ID NO. 50-68 and one or more of SEQ ID N 0.49, or SEQ ID N 0.98 116 and SEQ ID NO.

The EGFR gene mutation detecting liquid phase chip according to claim 1 or 2, wherein the specific primer for Exon 20 is: SEQ ID NO. 69-70. or SEQ ID N 0.117-118.

The EGFR gene mutation detecting liquid phase chip according to claim 1 or 2, wherein the specific primer for ΕΧΟΠ21 is: SEQ IDN 0.71 to 72, or SEQ ID NO. 119 to 120.

7. A specific primer for detecting EGFR gene mutation, wherein the specific primer is: a specific primer for the Exon 19 mutant, the specific primer is derived from one of SEQ ID NO. 26 to SEQ ID NO. 44, respectively. More than one base sequence, or one or more base sequences derived from the reverse complement sequence of SEQ ID NO. 74 - SEQ IDN 0.92, the 3' end of each specific primer is derived from the corresponding deletion region 3 'end adjacent 1 to 6 bases, and specifically recognize the corresponding mutation type, and a specific primer for Exon 19 wild type, which is derived from SEQ IDN 0.25 or its reverse complement a base sequence, and its 3' end contains a unique deletion base in the deletion region of each mutant to be detected, and is different from the specific primers of all mutants; or against Exon20, derived from SEQIDN0. a base sequence in 45~SEQID N0.46 or a reverse complement thereof; and/or a base sequence derived from Exon 21, derived from SEQ ID N0.47 to SEQ ID N0.48 or a reverse complement thereof; Specific primers for Exon 20 and Exon 21 3 'a base end of the last three bases of the mutation site corresponding to the wild type or site; all of these specific primers Tm value between 52 ~ 58 ° C.

The specific primer for detecting EGFR gene mutation according to claim 7, wherein the specific primer for the Exon 19 mutant has the 3' end of each specific primer derived from the corresponding deletion region. 1 to 3 bases adjacent to the 3' end.

The specific primer for detecting EGFR gene mutation according to claim 7, wherein the specific primer for Exon 19 is: one or more of SEQ ID NO. 50 to 68 and SEQ ID N 0.49, or SEQ More than one of ID N0.98 116 and SEQ ID NO.97.

The specific primer for detecting EGFR gene mutation according to claim 7, wherein the specific primer for Exon 20 is: SEQ ID NO. 69 to 70, or SEQ ID N 0.117 118; and/or Specific primers for Exon 21 are: SEQ ID N 0.71 to 72, or SEQ ID NO. 119 to 120.