CN103805684B

CN103805684B - The PCR primer that EML4-ALK fusion gene detects, test kit and liquid-phase chip

Info

Publication number: CN103805684B
Application number: CN201210447691.0A
Authority: CN
Inventors: 陈昌华; 陈菲; 许昌有
Original assignee: Surexam Bio Tech Co Ltd
Current assignee: Surexam Bio Tech Co Ltd
Priority date: 2012-11-09
Filing date: 2012-11-09
Publication date: 2016-04-27
Anticipated expiration: 2032-11-09
Also published as: CN103805684A; HK1180500A1

Abstract

The invention discloses a kind of PCR primer, test kit and the liquid-phase chip that detect EML4-ALK fusion gene and detect, described liquid-phase chip comprises: pcr amplification primer; The ASPE primer be made up of tag sequence and Auele Specific Primer, described primer sequence: SEQID NO.51, SEQ ID NO.52, SEQ ID NO.53, SEQ ID NO.54, SEQ ID NO.55, SEQ ID NO.56, SEQ ID NO.57, SEQ ID NO.58, SEQ ID NO.59, SEQ ID NO.60, SEQ ID NO.61, SEQ ID NO.62, SEQ ID NO.63, SEQ ID NO.64 and/or SEQ ID NO.65-71; Microballoon.Liquid-phase chip signal-noise ratio of the present invention is good, a step can complete the amplification that 21 kinds merge hypotype, and primer specificity is good.

Description

The PCR primer that EML4-ALK fusion gene detects, test kit and liquid-phase chip

Technical field

The invention belongs to biology field, relate to medical science and biotechnology, particularly relate to PCR primer, test kit and liquid-phase chip that a kind of EML4-ALK fusion gene detects.

Technical background

Chromosome rearrangement in cancer usually causes two, and independently gene fusion is together.In some cases, two gene coding regions link together, and cause the expression of fusion rotein, are called gene fusion (Genefusions).Gene fusion phenomenon is found in multiple blood and soft-tissue cancers the earliest, as leukemia, lymphoma etc.Within 2007, Japanese Scientists detects by the inversion of karyomit(e) 2p first in nonsmall-cell lung cancer (NSCLC) patient Postoperative Specimen, cause echinoderms microtubule associated class albumen 4(EML4) N-of the 13rd exon end and ALK(Nucleophosmin-anaplastic lymphoma kinase) the kinases district of the 20th exon merges, after testing, the expression product of this kind of fusion gene is chimeric Tyrosylprotein kinase, can in cell after spontaneous formation polymer, the promotion cell proliferation continued, leads oncogenic generation and transfer.And utilize the kinase whose inhibitor of ALK to limit its activity, the continuation of cancer cells effectively can be stoped to divide, and promote the apoptosis of cancer cells.Clinical trial results also shows, and merges positive patient use ALK inhibitor (as Crizotinib) treatment can extend the lifetime of patient to ALK.

(write a Chinese character in simplified form: E13 except EML4 gene the 13rd exon and ALK gene the 20th exon merge; A20), outside hypotype, in patients with lung cancer, there was reported other more than 20 plant the variation type merged.Analyze the topology discovery of these fusion genes, its ALK gene part all starts from 20 exons of tyrosine kinase domain in Codocyte, EML4 part then comprises the gene fragment of proteins encoded N end portion different in size after testing, and all EML4-ALK fusion genes all have biological function.

In general nonsmall-cell lung cancer crowd, EML4-ALK positive rate is lower, is about about 3% ~ 7%.At present, the ALK epidemiological study display that China is preliminary, the incidence that fusion gene detects is 11.6%, is 16.13% (10/62) in the incidence of gland cancer, be 19.23% (10/52) non-smoker, up to 42.80% in the gland cancer without EGFR or KRAS.

The existing launch detecting EML4-ALK gene fusion at present, as the VysisALKBreakApartFISHProbeKit series product of Abbott company, it analyzes chromosomal crack conditions based on fluorescence in situ hybridization technique (FISH), but whether it can only detect fusion gene exists, can not accurately draw concrete fused type, and susceptibility is lower; Traditional RT-PCR is sequencing, semiquantitive PCR technology then, then there is sensitivity low, sample easily pollutes, shortcoming that false positive rate is high, and regular-PCR method and quantitative fluorescent PCR all can not meet the needs of practical application due to the limitation detecting flux.

Summary of the invention

An object of the present invention is to provide a kind of PCR primer detecting EML4-ALK fusion gene, and described PCR primer can be used for separately or 21 kinds of gene fusion hypotype: E13 of parallel detection EML4-ALK fusion gene; A20, E13; Ins69; A20, E20; A20, E20; Ins18; A20, E6; A20, E6; Ins33; A20, E6; Ins18; A20, E2; A20, E2; Ins117; A20, E15; A20, E17; Ins29; A20, E17; Ins94; A20, E17; A20, E17; Ins68; A20, E18; A20, E21; A20, E14; Ins11; Del49A20, E14; Del14A20, E14; Ins64; Del69A20, E13; A21, E2; A21.

The technical scheme realizing above-mentioned purpose is as follows:

Detect a PCR primer for EML4-ALK fusion gene, include for fused type E13; A20, E13; Ins69; A20, E14; Ins11; Del49A20, E14; Del14A20, E14; Ins64; SEQIDNO.1 and SEQIDNO.3 of del69A20, for fused type E20; A20, E20; Ins18; A20, E21; SEQIDNO.1 and SEQIDNO.4 of A20, for fused type E6; A20, E6; Ins33; A20, E6; Ins18; SEQIDNO.1 and SEQIDNO.5 of A20, for fused type E2; A20, E2; Ins117; SEQIDNO.1 and SEQIDNO.6 of A20, for fused type E15; SEQIDNO.1 and SEQIDNO.7 of A20, the E17 for fused type; Ins29; A20, E17; Ins94; A20, E17; A20, E17; Ins68; A20, E18; SEQIDNO.1 and SEQIDNO.8 of A20, for fused type E13; SEQIDNO.2 and SEQIDNO.3 of A21 and/or for fused type E2; SEQIDNO.2 and SEQIDNO.6 of A21.

Another object of the present invention is to provide a kind of PCR kit detecting EML4-ALK fusion gene.

The technical scheme realizing above-mentioned purpose is as follows:

Detect a PCR kit for EML4-ALK fusion gene, include above-mentioned described PCR primer.

Wherein in an embodiment, described test kit also includes positive reference substance, and described positive reference substance is: for fused type E13; A20, E13; Ins69; A20, E14; Ins11; Del49A20, E14; Del14A20, E14; Ins64; The nucleic acid fragment containing SEQIDNO.1 and the sequence of matching with SEQIDNO.3 reverse complemental of del69A20, for fused type E20; A20, E20; Ins18; A20, E21; The nucleic acid fragment containing SEQIDNO.1 and the sequence of matching with SEQIDNO.4 reverse complemental of A20, for fused type E6; A20, E6; Ins33; A20, E6; Ins18; The nucleic acid fragment containing SEQIDNO.1 and the sequence of matching with SEQIDNO.5 reverse complemental of A20, for fused type E2; A20, E2; Ins117; The nucleic acid fragment containing SEQIDNO.1 and the sequence of matching with SEQIDNO.6 reverse complemental of A20, for fused type E15; The nucleic acid fragment containing SEQIDNO.1 and the sequence of matching with SEQIDNO.7 reverse complemental of A20, for fused type E17; Ins29; A20, E17; Ins94; A20, E17; A20, E17; Ins68; A20, E18; The nucleic acid fragment containing SEQIDNO.1 and the sequence of matching with SEQIDNO.8 reverse complemental of A20, for fused type E13; A21 containing SEQIDNO.2 and the nucleic acid fragment of sequence matched with SEQIDNO.3 reverse complemental and/or for fused type E2; A21 containing SEQIDNO.2 and the nucleic acid fragment of sequence that matches with SEQIDNO.6 reverse complemental.

Another object of the present invention is to provide a kind of liquid-phase chip detecting EML4-ALK fusion gene and detect, and described liquid-phase chip can be used for separately or 21 kinds of gene fusion hypotype: E13 of parallel detection EML4-ALK fusion gene; A20, E13; Ins69; A20, E20; A20, E20; Ins18; A20, E6; A20, E6; Ins33; A20, E6; Ins18; A20, E2; A20, E2; Ins117; A20, E15; A20, E17; Ins29; A20, E17; Ins94; A20, E17; A20, E17; Ins68; A20, E18; A20, E21; A20, E14; Ins11; Del49A20, E14; Del14A20, E14; Ins64; Del69A20, E13; A21, E2; A21.

The technical scheme realizing above-mentioned purpose is as follows:

The liquid-phase chip that EML4-ALK fusion gene detects, includes:

(A) above-mentioned described PCR primer;

(B). the ASPE primer for the different fused type of EML4-ALK fusion gene designs respectively: every bar ASPE primer holds the specific primer sequence for object fused type to form by 5 ' the tag sequence of holding and 3 ', and described specific primer sequence is: for E13; The SEQIDNO.51 of A20, for E13; Ins69; The SEQIDNO.52 of A20, for E20; The SEQIDNO.53 of A20, for E20; Ins18; The SEQIDNO.54 of A20, for E6; The SEQIDNO.55 of A20, for E6; Ins33; The SEQIDNO.56 of A20, for E6; Ins18; The SEQIDNO.57 of A20, for E2; The SEQIDNO.58 of A20, for E2; Ins117; The SEQIDNO.59 of A20, for E15; The SEQIDNO.60 of A20, for E17; Ins29; The SEQIDNO.61 of A20, for E17; Ins94; The SEQIDNO.62 of A20, for E17; The SEQIDNO.63 of A20, for E17; Ins68; The SEQIDNO.64 of A20, for E18; The SEQIDNO.65 of A20, for E21; The SEQIDNO.66 of A20, for E14; Ins11; The SEQIDNO.67 of del49A20, for E14; The SEQIDNO.68 of del14A20, for E14; Ins64; The SEQIDNO.69 of del69A20, for E13; The SEQIDNO.70 of A21 and/or for E2; The SEQIDNO.71 of A21; Described tag sequence is selected from SEQIDNO.30 ~ SEQIDNO.50;

(C). microballoon that have anti-tag sequence bag quilt, that have different colours coding, is also provided with spacer sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQIDNO.72 ~ SEQIDNO.92, and described anti-tag sequence can correspondingly be matched with selected tag complementary in (B).

Wherein in an embodiment, described ASPE primer is: for E13; The sequence be made up of SEQIDNO.30 and SEQIDNO.51 of A20, for E13; Ins69; The sequence be made up of SEQIDNO.31 and SEQIDNO.52 of A20, for E20; The sequence be made up of SEQIDNO.32 and SEQIDNO.53 of A20, for E20; Ins18; The sequence be made up of SEQIDNO.33 and SEQIDNO.54 of A20, for E6; The sequence be made up of SEQIDNO.34 and SEQIDNO.55 of A20, for E6; Ins33; The sequence be made up of SEQIDNO.35 and SEQIDNO.56 of A20, for E6; Ins18; The sequence be made up of SEQIDNO.36 and SEQIDNO.57 of A20, for E2; The sequence be made up of SEQIDNO.37 and SEQIDNO.58 of A20, for E2; Ins117; The sequence be made up of SEQIDNO.38 and SEQIDNO.59 of A20, for E15; The sequence be made up of SEQIDNO.39 and SEQIDNO.60 of A20, for E17; Ins29; The sequence be made up of SEQIDNO.40 and SEQIDNO.61 of A20, for E17; Ins94; The sequence be made up of SEQIDNO.41 and SEQIDNO.62 of A20, for E17; The sequence be made up of SEQIDNO.42 and SEQIDNO.63 of A20, for E17; Ins68; The sequence be made up of SEQIDNO.43 and SEQIDNO.64 of A20, for E18; The sequence be made up of SEQIDNO.44 and SEQIDNO.65 of A20, for E21; The sequence be made up of SEQIDNO.45 and SEQIDNO.66 of A20, for E14; Ins11; The sequence be made up of SEQIDNO.46 and SEQIDNO.67 of del49A20, for E14; The sequence be made up of SEQIDNO.47 and SEQIDNO.68 of del14A20, for E14; Ins64; The sequence be made up of SEQIDNO.48 and SEQIDNO.69 of del69A20, for E13; The sequence be made up of SEQIDNO.49 and SEQIDNO.70 of A21 and/or for E2; The sequence be made up of SEQIDNO.50 and SEQIDNO.71 of A21.

Major advantage of the present invention is:

1, EML4-ALK fusion gene provided by the present invention detects the identical rate of liquid phase chip reagent box and sequencing up to 100%.

2, detection liquid phase chip reagent box of the present invention uses step simple, use reverse transcription product as template, by the PCR primer of strictly screening, the amplification of the target sequence containing 21 kinds of EML4-ALK fusion gene hypotypes can be completed by One_step PCR, avoid the many uncertain factors existed in the complex operations processes such as repeated multiple times PCR, thus greatly can improve Detection accuracy, embody accurate qualitative analysis feature.

The liquid-phase chip of the EML4-ALK fusion gene 3, prepared by the present invention has extraordinary signal-noise ratio, substantially cross reaction is there is not between designed probe and anti-tag sequence, simultaneously, the ASPE type-special primer of the present invention's design has extraordinary specificity, accurately can distinguish the fusion gene hypotype of various type.

4, detection method of the present invention uses and merges the plasmid vector of subtype sequences as positive reference substance containing target detect, further ensures accuracy and the reliability of detection.

5, test kit provided by the present invention, the time required for its detection method well below conventional sequencing technologies, the needs of realistic especially application.

6, not only to overcome conventional solid chip susceptibility not high in the present invention, and the defect of the repeatability difference of detected result, improves existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value detected improves greatly, thus the sensitivity detected is further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.

Figure of description

Fig. 1 is the polyacrylamide gel electrophoresis picture of the group 1 of pcr amplification in embodiment 2;

Fig. 2 is the polyacrylamide gel electrophoresis picture of the group 2 of pcr amplification in embodiment 2;

Fig. 3 is the polyacrylamide gel electrophoresis picture of the group 3 of pcr amplification in embodiment 2.

Embodiment

Unaccounted ordinary method and condition in the embodiment of the present invention, the ordinary method according to those skilled in the art is carried out, and as " Molecular Cloning: A Laboratory guide " third edition, or implements according to the working method that manufacturer provides.

Embodiment 1 one kinds detects PCR primer and the positive reference substance of the detection of EML4-ALK fusion gene

One, RT-PCR amplification

1, reverse transcription

For the EML4-ALK fusion gene hypotype of various target detect, the present invention first uses random primer to carry out reverse transcription to target detect sample, mRNA reverse transcription in sample is become cDNA, specific experiment step, with reference to " Molecular Cloning: A Laboratory guide ", uses random primer to carry out the reverse transcription of mRNA for Conventional procedures.

2, pcr amplification

Fused type in table 1 is 21 kinds of EML4-ALK fusion gene hypotypes of target detect of the present invention, according to the nucleic acid composition characteristic of this fusion gene sequence, utilizes Primer5.0 to design forward primer and reverse primer.Use the cDNA of step 1 gained as template, according to the means (agarose electrophoresis, polyacrylamide gel electrophoresis, probe hybridization etc.) detected, increasing to 21 kinds of target detect fusion gene hypotypes, (the parallel amplification of multiple hypotype, is shown in embodiment 2; Or the parallel amplification of 21 kinds of hypotypes, is shown in embodiment 3, step 3,1, pcr amplification).

All primers are synthesized by Invitrogen company.Every bar primer after synthesis is mixed with the stock solution of 300pmol/mL respectively with 10mmol/LTrisBuffer.

Wherein, E13; A20 represents that the exons 13 of EML4 gene and the extron 20 of ALK gene merge.

Table 1EML4-ALK fusion gene hypotype pcr amplification primer

Two, positive reference substance

The present invention is according to the fusion sequence feature of 21 of target detect EML4-ALK fusion gene kinds of hypotypes, designing nucleic acid fragment is as the positive reference substance detected, the nucleic acid fragment of sequence that this positive reference substance is forward primer containing amplimer in table 1 and matches with reverse primer reverse complemental, nucleic acid fragment between the sequence of its forward primer and the pairing of reverse primer reverse complemental can be human genomic sequence, also can be derive from plant, the nucleotide sequence in the inhuman source such as microorganism, and the amplified production of the clip size of the synthesized positive reference substance target detect fused type corresponding to table 1 is in the same size, thus the effect realized in the positives contrast of amplification procedure, simultaneously, realize other Detection results (such as: when use derives from plant, during the nucleotide sequence in the inhuman source such as microorganism, human genomic sequence in positive reference substance can be avoided other to be detected to the positive pollution etc. of sample.）

In test kit of the present invention, for various target detect fusion gene hypotype, adopt and insert corresponding human genomic sequence as positive reference substance.Specifically in the SEQIDNO.9 ~ SEQIDNO.29 in table 2, trust money only intelligence biotechnology (Beijing) company limited carries out the synthesis of target insertion sequence, purified composition sequence is inserted on PMD18-T carrier, then be converted in bacillus coli DH 5 ɑ competent cell, screened by blue hickie, 21 kinds of EML4-ALK fusion gene hypotype recombinant plasmid dnas that establishing target detects are as positive reference substance.The 21 kinds of recombinant plasmids built are correct through two-way DNA sequencing qualification.Extract plasmid, ultraviolet spectrophotometer is quantitative, is diluted to 5.0 × 10 ¹⁰copies/ml is preserved in-20 ° of C.

The insertion sequence of table 2EML4-ALK fusion gene hypotype positive control plasmid

Sequence number	Fused type	Insert the subtype sequences of plasmid
			1	E13;A20	SEQ ID NO.9

2	E13;ins69;A20	SEQ ID NO.10
			3	E20;A20	SEQ ID NO.11
4	E20;ins18;A20	SEQ ID NO.12
			5	E6;A20	SEQ ID NO.13
6	E6;ins33;A20	SEQ ID NO.14
			7	E6;ins18;A20	SEQ ID NO.15
8	E2;A20	SEQ ID NO.16
			9	E2;ins117;A20	SEQ ID NO.17
10	E15;A20	SEQ ID NO.18
			11	E17;ins29;A20	SEQ ID NO.19
12	E17;ins94;A20	SEQ ID NO.20
			13	E17;A20	SEQ ID NO.21
14	E17;ins68;A20	SEQ ID NO.22
			15	E18;A20	SEQ ID NO.23
16	E21;A20	SEQ ID NO.24
			17	E14;ins11;del49A20	SEQ ID NO.25
18	E14;del14A20	SEQ ID NO.26
			19	E14;ins64;del69A20	SEQ ID NO.27
20	E13;A21	SEQ ID NO.28
			21	E2;A21	SEQ ID NO.29

Embodiment 2 uses the PCR primer in embodiment 1 to detect EML4-ALK fusion gene

1, pcr amplification

Utilize the pcr amplification primer in embodiment 1 described in table 1; pcr amplification is carried out to the mRNA reverse transcription product of target detect sample and positive control plasmid mixed solution; simultaneously; pcr amplification band is distinguished for the ease of using conventional electrophoretic technology; according to the size (as shown in table 1) of PCR primer; 21 of target detect kinds of fused type and corresponding positive control plasmid thereof are divided into 3 groups, carry out 3 independently pcr amplifications respectively simultaneously, and use polyacrylamide gel to detect.According to the size of amplified production, PCR primer is divided into required classification, and is detected by electrophoretic technique, belong to the ordinary method of art technology.This 3 group reaction system is respectively:

First PCR primer working fluid is prepared: according to 3 PCR groups in above table, respectively get primer stock solution 100ul corresponding in table 1 respectively in 1.5ml Eppendorf tube, mix, be 3 groups of multiple PCR primer working fluids.

In like manner, preparation positive control plasmid working fluid, according to the type of fusion gene in above table, is put into corresponding positive control plasmid in same 1.5ml Eppendorf tube, mixes, form positive control plasmid working fluid.

3 groups of multi-PRC reaction systems are all as follows:

Pcr amplification program is: 95 DEG C of 3min; 94 DEG C of 20s, 56 DEG C of 30s, 72 DEG C of 30s, 30 circulations; 72 DEG C of 10min; 4 DEG C save backup.

2, result detects and data analysis

Above-mentioned pcr amplification is used to detect 10 samples, in the pcr amplification product of this step, for the mRNA reverse transcription product of target detect sample, if there is the hypotype of certain fused type, then amplify respective strap, if the hypotype not containing certain fused type in sample, then can not amplify corresponding band, this amplified production detects by the ordinary method of the art technology such as sepharose, polyacrylamide gel, Southern hybridization, solid phase chip, liquid-phase chip, thus determines whether there is certain fusion gene hypotype.

Refer to Fig. 1 to Fig. 3, Fig. 1-Fig. 3 is respectively the electrophoresis picture that polyacrylamide gel detects amplified production.

The result of amplified production is detected from above-mentioned polyacrylamide gel electrophoresis, the amplimer specificity of detection method is high, for 21 kinds of fusion gene hypotypes, single band can be gone out by specific amplified, and the parallel detection of multiple fusion gene hypotype can be realized.

Embodiment 3 one kinds detects the liquid phase chip reagent box of EML4-ALK fusion gene

One, ASPE primer

For the E13 of target detect EML4-ALK gene; A20, E13; Ins69; A20, E20; A20, E20; Ins18; A20, E6; A20, E6; Ins33; A20, E6; Ins18; A20, E2; A20, E2; Ins117; A20, E15; A20, E17; Ins29; A20, E17; Ins94; A20, E17; A20, E17; Ins68; A20, E18; A20, E21; A20, E14; Ins11; Del49A20, E14; Del14A20, E14; Ins64; Del69A20, E13; A21, E2; A21 is totally 21 kinds of specific primer sequences merging hypotype and design.ASPE primer is made up of " Tag+ specific primer sequence ".ASPE primer sequence is as shown in the table:

Table 3ASPE primer sequence (Tag+ specific primer sequence)

Every bar ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence for anti-tag sequence on corresponding microballoon, and 3 ' end is saltant type or wild-type specific primer sequence (as shown in Table 3 above).All ASPE primers are synthesized by Invitrogen company.Every bar primer after synthesis is mixed with the stock solution of 100pmol/mL respectively with 10mmol/LTrisBuffer.

Two, the microballoon of anti-tag sequence bag quilt

According to designed ASPE specific primer sequence, select tag sequence, between the anti-tag sequence reducing each microballoon to greatest extent and tag and the ASPE specific primer sequence secondary structure that may be formed, the anti-tag sequence that two kinds of microballoons numberings of selection are corresponding on microballoon is as shown in table 2:

The anti-tag sequence that table 4 microballoon numbering is corresponding on microballoon

The 21 kinds of microballoon purchased from American Luminex companies selected, by anti-tag sequence bag by with on microballoon.Be connected with the spacerarm sequence of 5-10 T between anti-tag sequence and microballoon, before each anti-tag sequence, namely add the spacer sequence of the preceding paragraph 5-10 T, anti-tag sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.The anti-tag sequence sterilizing ddH2O of synthesis is made into the stock solution of 100nmol/ml.The process of microballoon bag quilt is as follows:

Get 5 × 10 respectively ⁶the carboxylated microballoon (purchased from Luminex company) of individual above-mentioned numbering is suspended in (pH4.5) in the MES solution of 50ul0.1mol/L, adds the anti-tag molecule (100nmol/ml) of 10ul synthesis.The EDC(N-(3-Dimethylaminopropyl of preparation 10ng/ml)-N-ethylcarbodiimide) (purchased from PierceChemical company) working fluid.The EDC working fluid of 2.5ul is added, constant-temperature incubation 30 minutes, then the EDC working fluid adding 2.5ul in microsphere suspensions, then constant-temperature incubation 30 minutes.After reaction terminates, the Tween-20 washing with 0.02% once, then is washed once with the SDS liquid of 0.1%.By washing after the microballoon being coated with anti-tag sequence be resuspended in 100ul Tris-EDTA solution [10mmol/LTris(pH8.0), in 1mmol/LEDTA, 2-8 DEG C keeps in Dark Place.

Three, from sample, amplify the primer that may there is fusion sequence

The invention provides a kind of liquid-phase chip detecting RT-PCR amplified production in embodiment 1, the test kit in embodiment 1 is used to carry out the parallel amplification of PCR to 21 of target detect kinds of fusion gene hypotypes and positive reference substance, concrete forward primer and reverse primer, as shown in table 1, concrete operation steps is shown in embodiment 2, use this embodiment, the parallel detection of 21 kinds of fusion gene hypotypes can be realized.

Embodiment 4 uses fusion gene in embodiment 3 to detect liquid-phase chip to the detection of sample

The formula of described various solution is as follows:

The MES damping fluid (pH5.0) formula (250ml) of 50mM:

2 × Tm hybridization buffer

Reagent	Source	Final concentration	The consumption of every 250ml
				1MTris-HCl，pH8.0	SigmaT3038	0.2M	50ml
5M NaCl	Sigma S5150	0.4M	20ml
				Triton X-100	Sigma T8787	0.16％	0.4ml

4 DEG C are stored in after filtration.

ExoSAP-IT test kit purchased from American USB company.

Biotin labeled dCTP is purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

One, the preparation of the RNA of sample

The extraction procedure of sample rna, is specifically shown in " Molecular Cloning: A Laboratory guide " third edition, and uses random primer to carry out reverse transcription to sample mRNA, see embodiment 1.

Two, the pcr amplification of testing sample

Use the detection method in embodiment 1, utilize the pcr amplification primer described in table 1, pcr amplification is carried out to the mRNA reverse transcription product of target detect sample and positive reference substance, realize the parallel amplification of the target sequence of 21 kinds of fusion gene hypotypes, one step amplifies 21 target sequences containing fusion gene hypotype, and product size is as shown in table 1.Primer sequence (SEQIDNO.1 ~ SEQIDNO.8) is shown in shown in above-mentioned table 1.

First PCR primer working fluid is prepared: the primer stock solution 100ul respectively getting SEQIDNO.1 ~ SEQIDNO.8 respectively, in 1.5ml Eppendorf tube, mixes and is multiple PCR primer working fluid.Multi-PRC reaction system is as follows:

Three, the enzyme of PCR primer cuts process

Illustrate with reference to ExoSAP-IT test kit, detailed step is as follows:

1. get the reacted product of 7.5ulPCR, add 3ulExoSAP-IT enzyme;

Hatch 15min for 2.37 DEG C.Hatch 15min for 80 DEG C, make unnecessary enzyme-deactivating.Product after enzyme cuts process is directly used in follow-up ASPE primer extension reaction.

Four, site-specific primer extension reaction (ASPE)

The site-specific primer utilizing table 3 to design carries out primer extension reaction, mixes biotin labeled dCTP in reaction process, thus makes biotin labelings multiple on reacted product band.

First prepare the ASPE primer working fluid of mixing: get each 10ul of corresponding ASPE primer stock solution in 1.5ml Eppendorf tube, add 10mmol/LTrisBuffer and mend to 200ul, mix and be ASPE mix primer working fluid.The system of ASPE reaction is as follows:

PCR program is: 96 DEG C of 2min; 94 DEG C of 30s, 58 DEG C of 1min, 72 DEG C of 2min, 30 circulations; 4 DEG C save backup.

Five, hybridization

1., according to the ASPE primer of design, (microballoon concentration is 2.5 × 10 to select 21 kinds of microballoons being coated with anti-tag sequence as described in Example 3 ⁵individual/ml).

2. get 1ul respectively and often plant the microballoon of numbering in the Eppendorf tube of 1.5ml;

3. microballoon is in >=centrifugal the 1-2min of 10000g;

4. supernatant discarded, microballoon is resuspended in 2 × Tm hybridization buffer of 100ul, and vortex mixes;

5. get the above-mentioned microsphere suspensions of 25ul in the 96 corresponding holes of hole filter plate, control wells adds the ddH of 25ul ₂o;

6. the ASPE reaction solution getting 5-25ul, in corresponding hole, uses ddH ₂o complements to 50ul;

7. encase 96 orifice plates with lucifuge with masking foil, 95 DEG C of 60s, 37 DEG C of 15min hatch hybridization;

8. the microballoon after hybridization is in >=centrifugal the 2-5min of 3000g;

9. remove supernatant, microballoon is resuspended in 1 × Tm hybridization buffer of 75ul;

10. microballoon is in >=centrifugal the 2-5min of 3000g;

Microballoon is resuspended in 1 × Tm hybridization buffer of 75ul by 11, adds the SA-PE (SA-PE) that 15ul concentration is 10ug/ml;

Hatch 15min for 12.37 DEG C, detect on Luminex instrument.

Six, result detects and data analysis

Reaction after product is detected by Luminex serial analysis instrument.Be greater than 100 for cut-off value with saltant type fluorescent value (MFI), when the MFI value that saltant type detects is greater than 100, judges that this sample exists this mutation type, otherwise judge that this sample is as corresponding wild-type.

Use present method to detect the EML4-ALK fusion gene hypotype of great amount of samples, detect with sequencing and compare with liquid-phase chip result, the RT-PCR product in embodiment is detected simultaneously, calculate the identical rate of method detected result provided by the present invention.Present method detect 20 increments this EML4-ALK fusion gene hypotype detected result and the sequencing result rate of coincideing reach 100%.Visible EML4-ALK fusion gene hypotype provided by the present invention detects liquid-phase chip can detect 21 kinds of EML4-ALK fusion gene hypotypes exactly, and result is reliable and stable.

One of table 5 pattern detection result (MFI)

Two of table 6 pattern detection result (MFI)

Three of table 7 pattern detection result (MFI)

Table 8 sample EML4-ALK analysis of fused genes result

Catalogue number(Cat.No.)	Liquid-phase chip detected result	Order-checking detected result
			11	Wild-type	Wild-type
12	Wild-type	Wild-type
			13	E18; A20 merges	E18; A20 merges
14	Wild-type	Wild-type
			15	Wild-type	Wild-type
16	Wild-type	Wild-type
			17	Wild-type	Wild-type
18	E20;ins18;A20	E20;ins18;A20
			19	Wild-type	Wild-type
20	Wild-type	Wild-type
			21	Wild-type	Wild-type
22	Wild-type	Wild-type
			23	Wild-type	Wild-type
24	Wild-type	Wild-type
			25	E17;A20	E17;A20
26	Wild-type	Wild-type
			27	Wild-type	Wild-type
28	Wild-type	Wild-type
			29	Wild-type	Wild-type
30	Wild-type	Wild-type

The selection of embodiment 5Tag sequence and Anti-Tag sequence

One, the design (selection of Tag sequence and Anti-Tag sequence) prepared of liquid-phase chip

With the E13 of EML4-ALK fusion gene; A20, E6; A20, E17; Ins29; A20, E17; Ins68; A20, E14; Ins11; Del49A20; E13; A21; E13; Ins69; A20; E20; A20; E6; Ins18; A20; E2; A20; E15; A20; E17; A20; E18; A20; E21; A20; E2; The detection liquid-phase chip of A21 hypotype is example, for the specific primer sequence (as shown in table 3) that this 15 kinds of fusion gene hypotype design ASPE primers 3 ' are held, the Tag sequence that ASPE primer 5 ' is held then is selected from SEQIDNO.30-SEQIDNO.50, accordingly, be coated in and microballoon is selected from SEQIDNO.72-SEQIDNO.92 with the anti-tag sequence that corresponding tag complementary matches.Specific design is as shown in following table (table 9).The synthesis of ASPE primer, anti-tag sequence bag by microballoon, amplimer, detection method etc. as described in embodiment 1, embodiment 2 and embodiment 3.

Design prepared by table 9 liquid-phase chip

Two, sample detection

Adopt liquid-phase chip prepared by above-mentioned design, detect sample 31-50 by testing process described in embodiment 2 and method, detected result is as follows:

One of table 10 pattern detection result (MFI)

Two of table 11 pattern detection result (MFI)

Three of table 12 pattern detection result (MFI)

Four of table 13 pattern detection result (MFI)

Five of table 14 pattern detection result (MFI)

Table 15 sample EML4-ALK fusion gene hypotype Analysis of test results

From above-described embodiment, other merges the liquid-phase chip of hypotype for difference, and ASPE primer uses different tag sequences, and its result is still reliable and stable, and concrete data are omitted.And ASPE primer is when selecting that in embodiment 3, tag sequence and specific primer sequence are arranged in pairs or groups, effect better (signal to noise ratio is better), see the present embodiment test group 1.Other different tag sequence and specific primer sequence are arranged in pairs or groups, and with coming to the same thing of embodiment 3,4 and the present embodiment, concrete data are omitted.

The cross reacting rate of the liquid-phase chip described in embodiment 6 embodiment 3 detects

One, experiment purpose

For fusion gene parallel detection liquid-phase chip of the present invention, carry out the detection of cross reacting rate.Get positive control plasmid, detect E13; A20, E13; Ins69; A20, E20; A20, E20; Ins18; A20, E6; A20, E6; Ins33; A20, E6; Ins18; A20, E2; A20, E2; Ins117; A20, E15; A20, E17; Ins29; A20, E17; Ins94; A20, E17; A20, E17; Ins68; A20, E18; A20, E21; A20, E14; Ins11; Del49A20, E14; Del14A20, E14; Ins64; Del69A20, E13; A21, E2; A21 totally 21 kinds merge the cross reacting rate of hypotypes.

Two, operation steps

1, prepare the mixed solution (being called for short: sample plasmid) of positive control plasmid, the concentration that this mixed solution contains often kind of plasmid is 106 copies/uL, loading 10uL.

2, carry out pcr amplification to the mixed solution of positive control plasmid, a step amplifies 21 target sequences containing fusion gene hypotype, and product size is as shown in table 1.

Preparation PCR primer working fluid: the primer stock solution 100ul respectively getting SEQIDNO.1 ~ SEQIDNO.8 respectively, in 1.5ml Eppendorf tube, mixes and is multiple PCR primer working fluid.Multi-PRC reaction system is as follows:

Three, the enzyme of PCR primer cuts process (as described in Example 4)

Four, site-specific primer extension reaction (as described in Example 4)

For 21 kinds of gene fusion hypotypes, get corresponding ASPE primer (as described in Example 3) respectively, be mixed with ASPE working fluid: get each 10ul of corresponding ASPE primer stock solution in 1.5ml Eppendorf tube, add 10mmol/LTrisBuffer to mend to 200ul, mix and be ASPE primer working fluid.Prepare 21 kinds of ASPE working fluids for different fused type respectively, and use following system, carry out ASPE extension respectively.The system of ASPE reaction is as follows:

Five, hybridization (as described in Example 4)

Six, result detects and data analysis

Reaction after product is detected by Luminex serial analysis instrument.

One of table 13 sample plasmids detection result ()

Table 14 sample plasmids detection result (two)

Table 15 sample plasmids detection result (three)

The selection of embodiment 7EML4-ALK fusion gene mutation detection specific primer sequence

One, the design (selection of wild-type and saltant type specific primer sequence) prepared of liquid-phase chip

With EML4-ALK fusion gene E20; A20; E6; Ins18; A20; E15; A20; E17; A20; E13; A21; E2; The detection liquid-phase chip of A21 hypotype is example, with the complementary sequence forward or backwards of this place, mutational site target sequence for template, respectively for E20; A20; E6; Ins18; A20; E15; A20; E17; A20; E13; A21; E2; The specific primer sequence that A21 hypotype design ASPE primer 3 ' is held, comprises preferred specific primer sequence in the embodiment of the present invention 1 and 2 alternative specific primer sequences, shown in table 16.

Table 16 specific primer sequence

With EML4-ALK fusion gene E20; A20; E6; Ins18; A20; E15; A20; E17; A20; E13; A21; E2; The detection liquid-phase chip of A21 hypotype is example, for E20; A20; E6; Ins18; A20; E15; A20; E17; A20; E13; A21; E2; A21 hypotype selects different specific primer sequences, and the tag sequence that ASPE primer 5 ' is held then is fixed as the best effect sequence in embodiment 1, and selects the anti-tag sequence corresponded, and specific design is as shown in following table (table 17).The synthesis of ASPE primer, anti-tag sequence bag by microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.

Design prepared by table 17 liquid-phase chip

Two, sample detection

Adopt liquid-phase chip prepared by above-mentioned design, detect sample 61-80 by testing process described in embodiment 2 and method, detected result is as follows:

One of table 18 pattern detection result (MFI)

Two of table 19 pattern detection result (MFI)

Table 20 sample EML4-ALK fusion gene hypotype Analysis of test results

Catalogue number(Cat.No.)	Liquid-phase chip detects knot	Order-checking detected result
			51	Wild-type	Wild-type
52	Wild-type	Wild-type
			53	Wild-type	Wild-type
54	Wild-type	Wild-type
			55	E2; A21 merges	E2; A21 merges
56	Wild-type	Wild-type
			57	Wild-type	Wild-type
58	Wild-type	Wild-type
			59	Wild-type	Wild-type
60	E20; A20 merges	E20; A20 merges
			61	Wild-type	Wild-type
62	Wild-type	Wild-type
			63	Wild-type	Wild-type
64	Wild-type	Wild-type
			65	Wild-type	Wild-type
66	Wild-type	Wild-type
			67	Wild-type	Wild-type
68	Wild-type	Wild-type
			69	Wild-type	Wild-type
70	Wild-type	Wild-type

From the present embodiment, when ASPE primer selects that in embodiment 3, specific primer sequence and tag sequence are arranged in pairs or groups, effect better (signal to noise ratio is better), see the present embodiment test group 4.Other derives from the different specific primer sequence of the complementary sequence forward or backwards of place, target detect site sequence and tag sequence is arranged in pairs or groups, with coming to the same thing of embodiment 4 and the present embodiment, namely be still that the specific primer sequence described in embodiment 3 is better from different tag sequence arranging effects, concrete data are omitted.

The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims

1. detect a PCR primer for EML4-ALK fusion gene, it is characterized in that, described PCR primer is: for E13; A20, E13; Ins69; A20, E14; Ins11; Del49A20, E14; Del14A20, E14; Ins64; SEQIDNO.1 and SEQIDNO.3 of del69A20, for E20; A20, E20; Ins18; A20, E21; SEQIDNO.1 and SEQIDNO.4 of A20, for E6; A20, E6; Ins33; A20, E6; Ins18; SEQIDNO.1 and SEQIDNO.5 of A20, for E2; A20, E2; Ins117; SEQIDNO.1 and SEQIDNO.6 of A20, for E15; SEQIDNO.1 and SEQIDNO.7 of A20, for E17; Ins29; A20, E17; Ins94; A20, E17; A20, E17; Ins68; A20, E18; SEQIDNO.1 and SEQIDNO.8 of A20, for E13; SEQIDNO.2 and SEQIDNO.3 of A21 and/or for E2; SEQIDNO.2 and SEQIDNO.6 of A21.

2. detect a PCR kit for EML4-ALK fusion gene, it is characterized in that, include PCR primer according to claim 1.

3. test kit according to claim 2, is characterized in that, also includes positive reference substance, and described positive reference substance is: for fused type E13; The SEQIDNO.9 of A20, for fused type E13; Ins69; The SEQIDNO.10 of A20, for fused type E20; The SEQIDNO.11 of A20, for fused type E20; Ins18; The SEQIDNO.12 of A20, for fused type E6; The SEQIDNO.13 of A20, for fused type E6; Ins33; The SEQIDNO.14 of A20, for fused type E6; Ins18; The SEQIDNO.15 of A20, for fused type E2; The SEQIDNO.16 of A20, for fused type E2; Ins117; The SEQIDNO.17 of A20, for fused type E15; The SEQIDNO.18 of A20, for fused type E17; Ins29; The SEQIDNO.19 of A20, for fused type E17; Ins94; The SEQIDNO.20 of A20, for fused type E17; The SEQIDNO.21 of A20, for fused type E17; Ins68; The SEQIDNO.22 of A20, for fused type E18; The SEQIDNO.23 of A20, for fused type E21; The SEQIDNO.24 of A20, for fused type E14; Ins11; The SEQIDNO.25 of del49A20, for fused type E14; The SEQIDNO.26 of del14A20, for fused type E14; Ins64; The SEQIDNO.27 of del69A20, for fused type E13; The SEQIDNO.28 of A21 and/or for fused type E2; The SEQIDNO.29 of A21.

4. a liquid-phase chip for EML4-ALK fusion gene detection, is characterized in that, include:

(A). PCR primer according to claim 1;

(B) for the ASPE primer that the different fused type of EML4-ALK fusion gene designs respectively: every bar ASPE primer holds the specific primer sequence for object fused type to form by 5 ' the tag sequence of holding and 3 ', and described specific primer sequence is: for E13; The SEQIDNO.51 of A20, for E13; Ins69; The SEQIDNO.52 of A20, for E20; The SEQIDNO.53 of A20, for E20; Ins18; The SEQIDNO.54 of A20, for E6; The SEQIDNO.55 of A20, for E6; Ins33; The SEQIDNO.56 of A20, for E6; Ins18; The SEQIDNO.57 of A20, for E2; The SEQIDNO.58 of A20, for E2; Ins117; The SEQIDNO.59 of A20, for E15; The SEQIDNO.60 of A20, for E17; Ins29; The SEQIDNO.61 of A20, for E17; Ins94; The SEQIDNO.62 of A20, for E17; The SEQIDNO.63 of A20, for E17; Ins68; The SEQIDNO.64 of A20, for E18; The SEQIDNO.65 of A20, for E21; The SEQIDNO.66 of A20, for E14; Ins11; The SEQIDNO.67 of del49A20, for E14; The SEQIDNO.68 of del14A20, for E14; Ins64; The SEQIDNO.69 of del69A20, for E13; The SEQIDNO.70 of A21 and/or for E2; The SEQIDNO.71 of A21; Described tag sequence is selected from SEQIDNO.30 ~ SEQIDNO.50;

5. the liquid-phase chip of EML4-ALK fusion gene detection according to claim 4, it is characterized in that, described ASPE primer is: for E13; The sequence be made up of SEQIDNO.30 and SEQIDNO.51 of A20, for E13; Ins69; The sequence be made up of SEQIDNO.31 and SEQIDNO.52 of A20, for E20; The sequence be made up of SEQIDNO.32 and SEQIDNO.53 of A20, for E20; Ins18; The sequence be made up of SEQIDNO.33 and SEQIDNO.54 of A20, for E6; The sequence be made up of SEQIDNO.34 and SEQIDNO.55 of A20, for E6; Ins33; The sequence be made up of SEQIDNO.35 and SEQIDNO.56 of A20, for E6; Ins18; The sequence be made up of SEQIDNO.36 and SEQIDNO.57 of A20, for E2; The sequence be made up of SEQIDNO.37 and SEQIDNO.58 of A20, for E2; Ins117; The sequence be made up of SEQIDNO.38 and SEQIDNO.59 of A20, for E15; The sequence be made up of SEQIDNO.39 and SEQIDNO.60 of A20, for E17; Ins29; The sequence be made up of SEQIDNO.40 and SEQIDNO.61 of A20, for E17; Ins94; The sequence be made up of SEQIDNO.41 and SEQIDNO.62 of A20, for E17; The sequence be made up of SEQIDNO.42 and SEQIDNO.63 of A20, for E17; Ins68; The sequence be made up of SEQIDNO.43 and SEQIDNO.64 of A20, for E18; The sequence be made up of SEQIDNO.44 and SEQIDNO.65 of A20, for E21; The sequence be made up of SEQIDNO.45 and SEQIDNO.66 of A20, for E14; Ins11; The sequence be made up of SEQIDNO.46 and SEQIDNO.67 of del49A20, for E14; The sequence be made up of SEQIDNO.47 and SEQIDNO.68 of del14A20, for E14; Ins64; The sequence be made up of SEQIDNO.48 and SEQIDNO.69 of del69A20, for E13; The sequence be made up of SEQIDNO.49 and SEQIDNO.70 of A21 and/or for E2; The sequence be made up of SEQIDNO.50 and SEQIDNO.71 of A21.

6. the liquid-phase chip that the EML4-ALK fusion gene according to claim 4 or 5 detects, it is characterized in that, described spacerarm is 5-10 T.