CN103805686A

CN103805686A - PCR (Polymerase Chain Reaction) primers, kit and liquid-phase chip for detecting ROS1 fused gene

Info

Publication number: CN103805686A
Application number: CN201210448618.5A
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: 2014-05-21
Anticipated expiration: 2032-11-09
Also published as: CN103805686B

Abstract

The invention discloses PCR (Polymerase Chain Reaction) primers, a kit and a liquid-phase chip for detecting an ROS1 fused gene. The liquid-phase chip comprises PCR amplification primers, an ASPE primer and a micro-ball, wherein the ASPE primer is composed of a tag sequence and specific primers; the sequences of the specific primers are as follows: a probe pair C6; SEQ ID NO.34 of R32 and the probe pair C6; SEQ ID NO.35 of R34 and a probe pair C5; SEQ ID NO.36 of the R34 and a probe pair S2; SEQ ID NO.37 of the R32 and a probe pair S4; SEQ ID NO.38 of the R32 and a probe pair S4; SEQ ID NO.39 of the R34 and a probe pair SL13; SEQ ID NO.40 of the R32 and a probe pair SL4; SEQ ID NO.41 of the R34 and a probe pair E10; SEQ ID NO.42 of the R34 and a probe pair L16; SEQ ID NO.43 of R35 and/or a probe pair T5; SEQ ID NO.44 of the R35. The liquid-phase chip has a very good signal-noise ratio and can finish the amplification of 11 fused subtypes in one step; the specific primer has very good specificity.

Description

PCR primer, test kit and liquid-phase chip that ROS1 fusion gene detects

Technical field

The invention belongs to biology field, relate to medical science and biotechnology, the concrete ROS1 fusion gene that relates to detects PCR primer, test kit and liquid-phase chip.

Technical background

V-ros UR2 sarcoma virus oncogene homologue 1(v-ros avian UR2sarcoma viral oncogene homolog 1, ROS1), within 1987, Rabin M. clones and v-ros sarcoma growth related gene, and called after ROS gene.ROS1 gene is the 32nd, and 34 and 35 exon places exist breakpoint, can merge with several genes.ROS1 fusion gene is proved to be new lung cancer and drives gene, in the formation of adenocarcinoma of lung, is bringing into play significant role.In the annual meeting of the 48th American Society of Clinical Oncology (ASCO) holding in June, 2012, the Shaw of masschusetts, u.s.a hospital general reported first about lung cancer recruit target spot ROS1 gene fusion patient's I clinical trial phase, clinical trial proves that it is the lung cancer molecular isoform that a class is new that ROS1 merges, and medicine gram azoles is very effective to this type of lung cancer for Buddhist nun (crizotinib).The present invention mainly detects and has done research with regard to ROS1 fusion gene.

What at present, the fusion gene research relevant to ROS1 was more mainly contains CD74-ROS1., SDC4-ROS1, SLC34A2-ROS1, EZR-ROS1, LRIG3-ROS1 and TPM3-ROS1 fusion gene.V1) CD74-ROS1 fusion gene

CD74 is MHC-II quasi-molecule associated constant chain (major histocompatibility complex II-associated invariantchain, Ii), mainly be expressed in antigen presenting cell (the antigen presentingcells such as dendritic cell, mononuclear macrophage, B cell, APC), it is the accessory molecule that is joined together to form nine aggressiveness in endoplasmic reticulum with new synthetic MHC-II quasi-molecule, belong to II type membrane molecule, relevant with antigen presentation function.Research also finds, CD74 molecule is also at endotheliocyte, tumor cells expression, with the generation of corresponding disease, develop relevant.Vascular endothelial cell is the monolayer cell that is positioned at blood vessel, has secretion vaso-active substance, participates in inflammatory cell and immunocyte migration, maintains many-sided biological functions such as vasomotion.At present research shows, the 6th exon of CD74 gene easily merges with ROS1 gene, and the incidence of this fusion gene detection is 1%.CD74-ROS1 fusion gene has susceptibility to gram azoles for Buddhist nun (crizotinib) medicine.

2) SDC4-ROS1 fusion gene

Syndecan-4(Syndecan-4 is called for short SDC4) formed by Suleparoid and chondroitin sulfate chain, belong to transmembrane protein glycan family, can be combined and serve as coreceptor with multiple heparin class somatomedin, conditioning signal transduction, simultaneously, SDC4 is as cyto-architectural moiety, also participates in iuntercellular, cell sticking with between extracellular matrix, somatomedin is combined and the behavior such as activation, macromolecular substance transhipment, immunne response.The promoter action that SDC4 forms keloid is expressed higher than normal skin tissue in keloid, and this high expression level promotes fibroblastic propagation in keloid.At present research shows, SDC4 gene the 2nd, 4 exons easily and ROS1 gene merge.

3) SLC34A2-ROS1 fusion gene

Solute Transport protein family 34 member 2(solute carrier family 34member2, SLC34A2) gene is positioned on 4p15.31-p15.2.SLC34A2 belongs to one of member of Solute Transport protein family SLC34, strongly expressed in lung, and the Pi translocator NaPi-IIb that coding Na relies on plays an important role in the balance that maintains inorganic phosphorus in body.High density dexamethasone promotes the SLC34A2 genetic expression of people's alveolar epithelial cells and calcium, the phosphorus transporter of extracellular fluid.At present, in Lines sample, detected SLC34A2-ROS1 fusion gene.Research finds, SLC34A2 gene the 4th, 13 exons easily and ROS1 gene merge.

4) EZR-ROS1, LRIG3-ROS1 and TPM3-ROS1 fusion gene

One of Ezrin albumen is ERM(Ezrin-Radixin-Moesin) family member, mainly participate in being connected between cytoskeleton and after birth.Studies have shown that, ezrin gene is crossed and is expressed in the esophageal carcinoma, carcinoma of the pancreas, carcinoma of endometrium, prostate cancer and other kinds of tumor cells.Be rich in leucine tumor-necrosis factor glycoproteins immunoglobulin-like albumen 3(leucine-rich repeats andimmunoglobin-like domains 3, LRIG3) be one of LRIG gene family member, in kinds of tumors, all there is down-regulated expression.Studies have shown that, LRIG3 gene overexpression can reduce invasion and attack and the transfer ability of glioma cell, accelerates apoptosis, suppresses generation and the development of glioma.Tropomyosin 3 genes (tropomyosin 3, TPM3) be one of albumen regulatory gene important in Muscle contraction process, it exists with Actin muscle duplex is parallel in myofiber filament, can affect and regulate and control the interaction between actomyosin.TPM3 transgenation is unable relevant with wire myopathy and skeletal muscle.Research shows, EZR, LRIG3 and TPM3 gene can both merge with ROS1 gene, have been found that at present, EZR gene exon10 and ROS1 gene the 34th exon merge, LRIG3 gene the 16th exon and ROS1 gene the 35th exon merge, and TPM3 gene the 5th exon and ROS1 gene the 35th exon merge.

At present, detect for most of RT-PCR method and the on-radiation coordination hybridization FISH method of adopting of research of RET fusion gene.RT-PCR method is to design primer for type and the amalgamation mode of known RET fusion gene, RNA reverse transcription is obtained after cDNA, mode by pcr amplification object fragment detects, and exists sensitivity low, the shortcoming that sample easily pollutes, false positive rate is high.Although but not radioactive in situ hybridization technology FISH method is comparatively directly perceived, but process of the test is too loaded down with trivial details, the reagent type needing is various, waste time and energy, and whether it can only detect fusion gene and exist, can not accurately draw concrete fused type, susceptibility is lower, thereby has limited to a certain extent the application of this method.

Summary of the invention

One of object of the present invention is to provide a kind of PCR primer of the ROS1 of detection fusion gene, and described PCR primer can be used for separately or the C6 of 11 kinds of gene fusion hypotype: CD74-ROS1 of parallel detection ROS1 fusion gene; R32, C6; R34, C5; R34; The S2 of SDC4-ROS1; R32, S4; R32, S4; R34; The SL13 of SLC34A2-ROS1; R32, SL4; R34; The E10 of EZR-ROS1; R34; The L16 of LRIG3-ROS1; The T5 of R35 and TPM3-ROS1; R35.

The technical scheme that realizes above-mentioned purpose is as follows:

Detect a PCR primer for ROS1 fusion gene, for C6; The SEQ ID NO.1 of R32 and SEQ ID NO.5, for C6; R34, C5; The SEQ ID NO.2 of R34 and SEQ ID NO.5, for S2; R32, S4; The SEQ ID NO.1 of R32 and SEQID NO.6, for S4; The SEQ ID NO.2 of R34 and SEQ ID NO.6, for SL13; The SEQ ID NO.1 of R32 and SEQ IDNO.7, for SL4; The SEQ ID NO.3 of R34 and SEQ ID NO.8, for E10; The SEQ ID NO.4 of R34 and SEQ IDNO.9, for L16; The SEQ ID NO.4 of R35 and SEQ ID NO.10 and/or for T5; The SEQ ID NO.4 of R35 and SEQID NO.11.

Another object of the present invention is to provide a kind of PCR test kit of the ROS1 of detection fusion gene.

The technical scheme that realizes this object is as follows:

Detect a PCR test kit for ROS1 fusion gene, include above-mentioned PCR primer.

In an embodiment, described PCR test kit also includes positive reference substance therein, and described positive reference substance is: for C6; R32's contains SEQ ID NO.1 and the nucleic acid fragment with the sequence of SEQ ID NO.5 reverse complemental pairing, for C6; R34, C5; R34 contain SEQ ID NO.2 and with the nucleic acid fragment of the sequence of SEQ ID NO.5 reverse complemental pairing, for S2; R32, S4; R32 contain SEQ ID NO.1 and with the nucleic acid fragment of the sequence of SEQ ID NO.6 reverse complemental pairing, for S4; R34 contain SEQ ID NO.2 and with the nucleic acid fragment of the sequence of SEQ ID NO.6 reverse complemental pairing, for SL13; R32 contain SEQ ID NO.1 and with the nucleic acid fragment of the sequence of SEQ ID NO.7 reverse complemental pairing, for SL4; R34 contain SEQ ID NO.3 and with the nucleic acid fragment of the sequence of SEQ ID NO.8 reverse complemental pairing, for E10; R34 contain SEQ ID NO.4 and with the nucleic acid fragment of the sequence of SEQ ID NO.9 reverse complemental pairing, for L16; R35 contain SEQ ID NO.4 and with the nucleic acid fragment of the sequence of SEQ ID NO.10 reverse complemental pairing and/or for T5; R35's contains SEQ ID NO.4 and the nucleic acid fragment with the sequence of SEQ ID NO.11 reverse complemental pairing.

More preferably, described positive reference substance is: for C6; The SEQ ID NO.12 of R32, for C6; The SEQ IDNO.13 of R34, for C5; The SEQ ID NO.14 of R34, for S2; The SEQ ID NO.15 of R32, for S4; The SEQ IDNO.16 of R32, for S4; The SEQ ID NO.17 of R34, for SL13; The SEQ ID NO.18 of R32, for SL4; The SEQ IDNO.19 of R34, for E10; The SEQ ID NO.20 of R34, for L16; The SEQ ID NO.21 of R35 and/or for T5; The SEQ ID NO.22 of R35.

Another object of the present invention is to provide a kind of liquid-phase chip of the ROS1 of detection fusion gene, and this liquid-phase chip can be realized independent or parallel qualitative detection to above-mentioned 11 kinds of ROS1 fusion gene hypotypes.

The technical scheme that realizes this object is as follows:

A liquid-phase chip that detects ROS1 fusion gene, includes:

(A) above-mentioned PCR primer;

(B). the ASPE primer designing respectively for the different fused type of ROS1 fusion gene: every ASPE primer is made up of for the specific primer sequence of object fused type tag sequence and the 3 ' end of 5 ' end, and described specific primer sequence is: for C6; The SEQ ID NO.34 of R32, for C6; The SEQ ID NO.35 of R34, for C5; The SEQ ID NO.36 of R34, for S2; The SEQ ID NO.37 of R32, for S4; The SEQ ID NO.38 of R32, for S4; The SEQ ID NO.39 of R34, for SL13; The SEQ ID NO.40 of R32, for SL4; The SEQ ID NO.41 of R34, for E10; The SEQ ID NO.42 of R34, for L16; The SEQ ID NO.43 of R35 and/or for T5; The SEQ ID NO.44 of R35; Described tag sequence is selected from SEQ ID NO.23-SEQ IDNO.33;

(C). there is microballoon that anti-tag sequence is coated with, that there is different colours coding, in the middle of described anti-tag sequence is connected with microballoon, be also provided with spacerarm sequence; Described anti-tag sequence is selected from SEQ ID NO.45-SEQ ID NO.55, and described anti-tag sequence can be correspondingly with (B) in selected tag sequence complementary pairing.

Another object of the present invention is to provide a kind of Auele Specific Primer for detection of ROS1 fusion gene.

Concrete technical scheme is as follows:

For detection of an Auele Specific Primer for ROS1 fusion gene, described Auele Specific Primer is: for C6; The SEQID NO.34 of R32, for C6; The SEQ ID NO.35 of R34, for C5; The SEQ ID NO.36 of R34, for S2; The SEQ ID NO.37 of R32, for S4; The SEQ ID NO.38 of R32, for S4; The SEQ ID NO.39 of R34, for SL13; The SEQ ID NO.40 of R32, for SL4; The SEQ ID NO.41 of R34, for E10; The SEQ ID NO.42 of R34, for L16; The SEQ ID NO.43 of R35 and/or for T5; The SEQ ID NO.44 of R35.

Major advantage of the present invention is:

1, the identical rate that ROS1 fusion gene provided by the present invention detects liquid-phase chip and sequencing is up to 100%.

2, detection liquid-phase chip step of the present invention is simple, use reverse transcription product as template, by the PCR primer of strict screening, can a step PCR complete the amplification of the target sequence that contains 11 kinds of ROS1 fusion gene hypotypes, the many uncertain factors that exist in the complex operations processes such as repeated multiple times PCR are avoided, thereby can greatly improve Detection accuracy, embody accurate qualitative analysis feature.

3, detection kit of the present invention is used the plasmid vector that contains target detect fusion hypotype sequence as positive reference substance, has further guaranteed the accuracy and the reliability that detect.

4, the liquid-phase chip of the prepared ROS1 fusion gene of the present invention has extraordinary signal-noise ratio, between designed probe and anti-tag sequence, substantially there is not cross reaction, simultaneously, the ASPE type specificity primer of the present invention's design has extraordinary specificity, can accurately distinguish the fusion gene hypotype of various types.

5, the needed time of detection method provided by the present invention is well below conventional sequencing technologies, the needs of realistic especially application.

6, not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the poor defect of repeatability of detected result is improved existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value detecting improves greatly, thereby the sensitivity detecting 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.

Embodiment

Unaccounted normal condition and method in the embodiment of the present invention, the ordinary method adopting according to those skilled in the art is carried out, and as " molecular cloning experiment guide " third edition, or the working method providing according to production firm is implemented.

1 one kinds of embodiment detect PCR primer and the positive reference substance that ROS1 fusion gene detects

One, RT-PCR amplification

1, reverse transcription

The ROS1 fusion gene hypotype detecting for all types of target, the present invention first uses random primer to carry out reverse transcription to target detect sample, mRNA reverse transcription in sample is become to cDNA, specific experiment step is with reference to " molecular cloning experiment guide ", and the reverse transcription that uses random primer to carry out mRNA is routine operation step.

2, pcr amplification

Fused type in table 1 is 11 kinds of ROS1 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 design forward primer and reverse primer.Use the cDNA of step 1 gained as template, to the amplification that walks abreast of 11 kinds of target detect fusion gene hypotypes.

All primers are synthesized by Invitrogen company.Every primer after synthetic is mixed with respectively the stock solution of 300pmol/mL with 10mmol/L Tris Buffer.

Wherein, C6; R32 represents that the exon 6 of CD74 gene and the exon 32 of ROS1 gene merge.

Table 1ROS1 fusion gene hypotype amplimer

Two, positive reference substance

The present invention is according to the fusion sequence feature of 11 kinds of hypotypes of target detect ROS1 fusion gene, designing nucleic acid fragment is as the positive reference substance detecting, this positive reference substance is the forward primer that contains amplimer in table 1 and the nucleic acid fragment with the sequence of reverse primer reverse complemental pairing, 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 to derive from plant, the nucleotide sequence in the inhuman source such as microorganism, and in the clip size of the positive reference substance of synthesized and table 1, the amplified production of corresponding target detect fused type is in the same size, thereby realize the effect of positive control in amplification procedure, simultaneously, (for example: when use derives from plant realize other experiment purpose, when the nucleotide sequence in the inhuman source such as microorganism, can avoid human genomic sequence in positive reference substance to detect the positive pollution etc. of sample to other.) in detection method of the present invention, detect fusion gene hypotype for all types of target, adopt and insert corresponding human genomic sequence as positive reference substance.Specifically in the SEQ ID NO.12 ~ SEQ ID NO.22 in table 2, trust money only intelligence biotechnology (Beijing) company limited is carried out the synthetic of target insertion sequence, purified composition sequence is inserted on PMD18-T carrier, then be converted in bacillus coli DH 5 ɑ competent cell, screen by blue hickie, 11 kinds of ROS1 fusion gene hypotype recombinant plasmid dnas that establishing target detects are as positive reference substance.11 kinds of recombinant plasmids that build are identified correct through two-way DNA sequencing.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 2ROS1 fusion gene hypotype positive control plasmid

Embodiment 2 uses the PCR primer in embodiment 1 to detect ROS1 fusion gene

1, pcr amplification

Utilize the pcr amplification primer described in table 1 in embodiment 1, mRNA reverse transcription product to target detect sample and positive control plasmid mixed solution carry out pcr amplification, simultaneously, distinguish pcr amplification band for the ease of using conventional electrophoretic technique, according to the size of PCR product (as shown in table 1), 11 kinds of fused type and the corresponding positive control plasmid thereof of target detect are divided into 2 groups, carry out respectively 2 independently pcr amplifications simultaneously, and use respectively sepharose and polyacrylamide gel to detect, according to the size of amplified production, PCR primer is divided into required classification, and detect by electrophoretic technique, belong to the ordinary method of art technology.This 2 group reaction system is respectively:

First prepare PCR primer working fluid: according to 2 PCR groups in above table, respectively get respectively primer stock solution 100ul corresponding in table 1 in 1.5ml Eppendorf tube, mix, be 2 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, and forms positive control plasmid working fluid.

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

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

2, result detects and data analysis

Use above-mentioned pcr amplification 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, amplify respective strap, if do not contain the hypotype of certain fused type in sample, can not amplify corresponding band, the ordinary method that this amplified production can pass through the art technology such as sepharose, polyacrylamide gel, Southern hybridization, solid phase chip, liquid-phase chip detects, thereby determines whether to exist certain fusion gene hypotype.Refer to Fig. 1 to Fig. 2, Fig. 1-Fig. 2 is respectively the electrophoresis picture of polyacrylamide gel detection amplified production.

Detected the result of amplified production from above-mentioned polyacrylamide gel electrophoresis, the amplimer specificity of detection method of the present invention is high, for 11 kinds of fusion gene hypotypes, can go out single band by specific amplified, and can realize the parallel detection of multiple fusion gene hypotype.

3 one kinds of embodiment detect the liquid-phase chip of ROS1 fusion gene

One, ASPE primer

For the C6 of the CD74-ROS1 of target detect ROS1 fusion gene; R32, C6; R34, C5; R34; The S2 of SDC4-ROS1; R32, S4; R32, S4; R34; The SL13 of SLC34A2-ROS1; R32, SL4; R34; The E10 of EZR-ROS1; R34; The L16 of LRIG3-ROS1; The T5 of R35 and TPM3-ROS1; R35 is the specific primer sequence of totally 11 kinds of fusion hypotypes 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 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 primer after synthetic is mixed with respectively the stock solution of 100pmol/mL with 10mmol/L Tris Buffer.

Two, the coated microballoon of anti-tag sequence

According to designed ASPE specific primer sequence, select tag sequence, reduce to greatest extent between the anti-tag sequence of each microballoon and secondary structure that tag and ASPE specific primer sequence may form, on 11 kinds of microballoons numberings of selection and microballoon, corresponding anti-tag sequence is as shown in table 4:

Corresponding anti-tag sequence on table 4 microballoon numbering and microballoon

11 kinds of microballoons selecting are purchased from Luminex company of the U.S., by coated anti-tag sequence and microballoon.Between anti-tag sequence and microballoon, be connected with the spacerarm order of 5-10 T, before each anti-tag sequence, add the spacerarm sequence of the preceding paragraph 5-10 T, anti-tag sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.By synthetic anti-tag sequence sterilizing ddH ₂o is made into the stock solution of 100nmol/ml.The coated process of microballoon is as follows:

Get respectively 5 × 10 ⁶the carboxylated microballoon (purchased from Luminex company) of individual above-mentioned numbering is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds the synthetic anti-tag molecule (100nmol/ml) of 10ul.The EDC(N-(3-Dimethylaminopropyl of preparation 10ng/ml)-N-ethylcarbodiimide) (purchased from Pierce Chemical company) working fluid.Toward the EDC working fluid that adds 2.5ul in microballoon suspension, constant-temperature incubation 30 minutes, then add the EDC working fluid of 2.5ul, then constant-temperature incubation 30 minutes.After reaction finishes, the Tween-20 with 0.02% washs once, then washs once with 0.1% SDS liquid.By washing after the microballoon that is coated with anti-tag sequence be resuspended in 100ul Tris-EDTA solution [10mmol/L Tris(pH8.0), in 1mmol/LEDTA, 2-8 ℃ keeps in Dark Place.

Three, from sample, amplify the primer that may have fusion sequence

The invention provides the liquid-phase chip of RT-PCR amplified production in a kind of embodiment of detection 1, use the test kit in embodiment 1 to carry out the parallel amplification of PCR to 11 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, can realize the parallel detection of 11 kinds of fusion gene hypotypes.

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

The formula of described various solution is as follows:

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
5MNaCl	Sigma?S5150	0.4M	20ml
				Triton?X-100	Sigma?T8787	0.16％	0.4ml

After filtration, be stored in 4 ℃.

ExoSAP-IT test kit is purchased from USB company of the U.S..

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 experiment guide " third edition, and uses random primer to carry out reverse transcription to sample mRNA, referring to 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, mRNA reverse transcription product and positive reference substance to target detect sample carry out pcr amplification, realize the parallel amplification of the target sequence of 11 kinds of fusion gene hypotypes, one step amplifies 11 target sequences that contain fusion gene hypotype, and product size is as shown in table 1.Primer sequence (SEQ ID NO.1 ~ SEQID NO.11) is shown in shown in above-mentioned table 1.

First prepare PCR primer working fluid: respectively get respectively the primer stock solution 100ul of SEQ ID NO.1 ~ SEQ ID NO.11 in 1.5ml Eppendorf tube, mix and be multiple PCR primer working fluid.Multi-PRC reaction system is as follows:

Three, the enzyme of PCR product is cut processing

With reference to the explanation of ExoSAP-IT test kit, detailed step is as follows:

1. get the reacted product of 7.5ul PCR, add 3ul ExoSAP-IT enzyme;

Hatch 15min for 2.37 ℃.Hatch 15min for 80 ℃, make unnecessary enzyme-deactivating.Enzyme is cut product after treatment and is directly used in follow-up ASPE primer extension reaction.

Four, site-specific primer extension reaction (ASPE)

Utilize the locus specificity primer that table 3 designs to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thereby make multiple biotin labeling on reacted product band.

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

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

Five, hybridization

1. according to the ASPE primer of design, (microballoon concentration is 2.5 × 10 to select as described in Example 3 11 kinds of microballoons that are coated with anti-tag sequence ⁵individual/ml).

2. get respectively the microballoon of every kind of numbering of 1ul 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 microballoon suspension of 25ul in the 96 corresponding holes of hole filter plate, control wells adds the ddH of 25ul ₂o;

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

7. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 37 ℃ of 15min are hatched 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;

11 are resuspended in microballoon in 1 × Tm hybridization buffer of 75ul, and adding 15ul concentration is the SA-PE (SA-PE) of 10ug/ml;

Hatch 15min for 12.37 ℃, on Luminex instrument, detect.

Six, result detects and data analysis

Reaction after product detects by Luminex serial analysis instrument.Be greater than 100 as cut-off values take saltant type fluorescent value (MFI), in the time that the MFI value of saltant type detection 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 ROS1 fusion gene hypotype of great amount of samples, detect with liquid-phase chip result and compare with sequencing, the RT-PCR product in embodiment 1 is detected simultaneously, calculate the identical rate of method detected result provided by the present invention.Present method detects 20 increments ROS1 fusion gene hypotype detected result and the sequencing result rate of coincideing originally and reaches 100%.Visible ROS1 fusion gene hypotype provided by the present invention detects liquid-phase chip can detect 11 kinds of ROS1 fusion gene hypotypes exactly, and result is reliable and stable.

One of table 5 pattern detection result (MFI)

Sample	C6;R32	C6;R34	C5;R34	S2;R32	S4;R32	S4;R34
							Positive control	4130	3380	4139	2864	3534	3782
Negative control	20	25	13	14	25	22
							11	22	42	18	20	43	29
12	31	39	21	17	47	23
							13	33	45	38	27	43	39
14	43	28	31	27	44	32
							15	21	33	20	20	40	37
16	37	33	33	20	3371	32
							17	35	28	3768	19	38	43
18	26	34	27	34	45	37
							19	25	45	30	36	29	37
20	21	35	15	21	32	29
							21	38	34	35	31	29	28
22	22	33	14	33	47	46
							23	40	35	19	20	40	36
24	24	42	14	32	47	31
							25	23	38	18	23	39	38
26	36	40	31	24	25	30
							27	43	47	23	21	32	36
28	43	39	38	35	37	42
							29	40	36	28	18	35	42
30	24	39	27	36	46	46

Two of table 6 pattern detection result (MFI)

Sample	SL13;R32	SL4;R34	E10;R34	L16;R35	T5;R35
						Positive control	2311	2648	4433	3998	2695
Negative control	11	17	12	18	21
						11	22	35	33	37	42
12	23	3416	27	26	32
						13	17	21	26	20	25
14	28	21	27	42	38

15	18	37	21	25	38
						16	27	31	32	35	24
17	28	26	33	27	43
						18	15	34	18	39	27
19	12	33	22	34	43
						20	23	23	17	19	36
21	27	27	17	30	33
						22	32	37	3740	19	29
23	31	22	18	39	21
						24	17	33	20	40	42
25	35	25	34	23	30
						26	22	21	32	23	44
27	15	32	17	19	34
						28	21	23	29	18	32
29	33	33	21	34	43
						30	13	26	14	40	44

Table 7 sample ROS1 analysis of fused genes result

Sample	Liquid-phase chip detected result	Order-checking detected result
			11	Wild-type	Wild-type
12	SL4; R34 merges	SL4; R34 merges
			13	Wild-type	Wild-type
14	Wild-type	Wild-type
			15	Wild-type	Wild-type
16	S4; R32 merges	S4; R32 merges
			17	C5; R34 merges	C5; R34 merges
18	Wild-type	Wild-type
			19	Wild-type	Wild-type
20	Wild-type	Wild-type
			21	Wild-type	Wild-type
22	E10; R34 merges	E10; R34 merges
			23	Wild-type	Wild-type
24	Wild-type	Wild-type

25	Wild-type	Wild-type
			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) that prepared by liquid-phase chip

With the C6 of ROS1 fusion gene; R32, S2; R32; SL13; R32; E10; R34; L16; R35 and T5; The detection liquid-phase chip of R35 hypotype is example, for the specific primer sequence (as shown in table 3) of these 6 kinds of fusion gene hypotype design ASPE primer 3 ' ends, the Tag sequence of ASPE primer 5 ' end is selected from SEQ ID NO.23-SEQ ID NO.33, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing being coated on microballoon is selected from SEQ ID NO.45-SEQ ID NO.55.Specific design is as shown in following table (table 8).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. as described in embodiment 1, embodiment 2 and embodiment 3.

Design prepared by table 8 liquid-phase chip

Two, sample detection

The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 31-50 by testing process described in embodiment 2 and method, and detected result is as follows:

One of table 9 pattern detection result (MFI)

Two of table 10 pattern detection result (MFI)

Table 11 sample ROS1 fusion gene hypotype Analysis of test results

Catalogue number(Cat.No.)	Liquid-phase chip detects knot	Order-checking detected result
			31	Wild-type	Wild-type
32	Wild-type	Wild-type
			33	Wild-type	Wild-type

34	Wild-type	Wild-type
			35	Wild-type	Wild-type
36	Wild-type	Wild-type
			37	Wild-type	Wild-type
38	Wild-type	Wild-type
			39	Wild-type	Wild-type
40	Wild-type	Wild-type
			41	Wild-type	Wild-type
42	Wild-type	Wild-type
			43	Wild-type	Wild-type
44	E10; R34 merges	E10; R34 merges
			45	Wild-type	Wild-type
46	Wild-type	Wild-type
			47	C6; R32 merges	C6; R32 merges
48	Wild-type	Wild-type
			49	Wild-type	Wild-type
50	Wild-type	Wild-type

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 while selecting in embodiment 3 collocation of tag sequence and specific primer sequence, effect better (signal to noise ratio is better), referring to the present embodiment test group 1.Other different tag sequences and specific primer sequence collocation, with coming to the same thing of embodiment 4 and the present embodiment, concrete data are omitted.

The cross reacting rate of embodiment 6 liquid-phase chips 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 the C6 of CD74-ROS1; R32, C6; R34, C5; R34; The S2 of SDC4-ROS1; R32, S4; R32, S4; R34; The SL13 of SLC34A2-ROS1; R32, SL4; R34; The E10 of EZR-ROS1; R34; The L16 of LRIG3-ROS1; The T5 of R35 and TPM3-ROS1; R35 is the cross reacting rate of totally 11 kinds of fusion hypotypes.

Two, operation steps

1, the mixed solution of preparation positive control plasmid (being called for short: sample plasmid), the concentration that this mixed solution contains every kind of plasmid is 10 ⁶copy/uL, loading 10uL, repeats 3 times.

2, the mixed solution of positive control plasmid is carried out to pcr amplification, a step amplifies 11 target sequences that contain fusion gene hypotype, and product size is as shown in table 1.

Preparation PCR primer working fluid: respectively get respectively the primer stock solution 100ul of SEQ ID NO.1 ~ SEQ ID NO.11 in 1.5ml Eppendorf tube, mix and be multiple PCR primer working fluid.Multi-PRC reaction system is as follows:

Three, the enzyme of PCR product is cut processing (as described in Example 4)

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

For 11 kinds of gene fusion hypotypes, get respectively corresponding ASPE primer, be mixed with ASPE working fluid: get the each 10ul of corresponding ASPE primer stock solution in 1.5ml Eppendorf tube, add 10mmol/L Tris Buffer to mend to 200ul, mix and be ASPE primer working fluid.Prepare respectively 11 kinds of ASPE working fluids for different fused type, and use following system, carry out respectively ASPE extension.The system of ASPE reaction is as follows:

Five, hybridization (as described in Example 4)

Six, result detects and data analysis

Reaction after product detects by Luminex serial analysis instrument.

Table 12 sample plasmid detected result

The selection of embodiment 7ROS1 fusion gene mutation detection specific primer sequence

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

With ROS1 fusion gene C6; R34; S4; R32; SL13; The detection liquid-phase chip of R32 hypotype is example, take the complementary sequence forward or backwards of this place, mutational site target sequence as template, respectively for C6; R34; S4; R32; SL13; The specific primer sequence of R32 hypotype design ASPE primer 3 ' end, comprises preferred specific primer sequence and 2 alternative specific primer sequences in the embodiment of the present invention 1, as shown in table 13.

Table 13 specific primer sequence

With ROS1 fusion gene C6; R34; S4; R32; SL13; The detection liquid-phase chip of R32 hypotype is example,, for C6; R34; S4; R32; SL13; R32 hypotype is selected different specific primer sequences, and the tag sequence of ASPE primer 5 ' end is fixed as the best effect sequence in embodiment 1, and selects the anti-tag sequence of answering in contrast, and specific design is as shown in following table (table 14).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. as described in embodiment 3 and embodiment 4.

Design prepared by table 14 liquid-phase chip

Two, sample detection

The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 51-70 by testing process described in embodiment 2 and method, and detected result is as follows:

Table 15 pattern detection result (MFI)

Table 16 sample ROS1 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	S4; R32 merges	S4; R32 merges

56	Wild-type	Wild-type
			57	Wild-type	Wild-type
58	Wild-type	Wild-type
			59	C6; R34 merges	C6; R34 merges
60	Wild-type	Wild-type
			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 is selected in embodiment 3 collocation of specific primer sequence and tag sequence, effect better (signal to noise ratio is better), referring to the present embodiment test group 4.Other derives from different specific primer sequences and the collocation of tag sequence of the complementary sequence forward or backwards of place, target detect site sequence, with coming to the same thing of embodiment 3 and the present embodiment, be still that the specific primer sequence described in embodiment 4 is better from different tag sequence arranging effects, concrete data are omitted.

The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore 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. a PCR primer that detects ROS1 fusion gene, is characterized in that, described PCR primer is for C6; The SEQID NO.1 of R32 and SEQ ID NO.5, for C6; R34, C5; The SEQ ID NO.2 of R34 and SEQ ID NO.5, for S2; R32, S4; The SEQ ID NO.1 of R32 and SEQ ID NO.6, for S4; The SEQ ID NO.2 of R34 and SEQ ID NO.6, for SL13; The SEQ ID NO.1 of R32 and SEQ ID NO.7, for SL4; The SEQ ID NO.3 of R34 and SEQ ID NO.8, for E10; The SEQ ID NO.4 of R34 and SEQ ID NO.9, for L16; The SEQ ID NO.4 of R35 and SEQ ID NO.10 and/or for T5; The SEQ ID NO.4 of R35 and SEQ ID NO.11.

2. a PCR test kit that detects ROS1 fusion gene, is characterized in that, includes PCR primer claimed in claim 1.

3. PCR test kit according to claim 2, is characterized in that, also includes positive reference substance, and described positive reference substance is: for C6; R32's contains SEQ ID NO.1 and the nucleic acid fragment with the sequence of SEQ ID NO.5 reverse complemental pairing, for C6; R34, C5; R34 contain SEQ ID NO.2 and with the nucleic acid fragment of the sequence of SEQ ID NO.5 reverse complemental pairing, for S2; R32, S4; R32 contain SEQ ID NO.1 and with the nucleic acid fragment of the sequence of SEQ ID NO.6 reverse complemental pairing, for S4; R34 contain SEQ ID NO.2 and with the nucleic acid fragment of the sequence of SEQ ID NO.6 reverse complemental pairing, for SL13; R32 contain SEQ ID NO.1 and with the nucleic acid fragment of the sequence of SEQ ID NO.7 reverse complemental pairing, for SL4; R34 contain SEQ ID NO.3 and with the nucleic acid fragment of the sequence of SEQ ID NO.8 reverse complemental pairing, for E10; R34 contain SEQ ID NO.4 and with the nucleic acid fragment of the sequence of SEQ ID NO.9 reverse complemental pairing, for L16; R35 contain SEQ ID NO.4 and with the nucleic acid fragment of the sequence of SEQ ID NO.10 reverse complemental pairing and/or for T5; R35's contains SEQ ID NO.4 and the nucleic acid fragment with the sequence of SEQ ID NO.11 reverse complemental pairing.

4. PCR test kit according to claim 2, is characterized in that, described positive reference substance is: for C6; The SEQ ID NO.12 of R32, for C6; The SEQ ID NO.13 of R34, for C5; The SEQ ID NO.14 of R34, for S2; The SEQ ID NO.15 of R32, for S4; The SEQ ID NO.16 of R32, for S4; The SEQ ID NO.17 of R34, for SL13; The SEQ ID NO.18 of R32, for SL4; The SEQ ID NO.19 of R34, for E10; The SEQ ID NO.20 of R34, for L16; The SEQ ID NO.21 of R35 and/or for T5; The SEQ ID NO.22 of R35.

5. a liquid-phase chip that detects ROS1 fusion gene, is characterized in that, includes:

(A) PCR primer claimed in claim 1;

6. require the liquid-phase chip described in 5 according to power, it is characterized in that, described ASPE primer is: for C6; The sequence being made up of SEQ IDNO.23 and SEQ ID NO.34 of R32, for C6; The sequence of the SEQ ID NO.24 of R34 and SEQ ID NO.35 composition, for C5; The sequence of the SEQ ID NO.25 of R34 and SEQ ID NO.36 composition, for S2; The sequence of the SEQ ID NO.26 of R32 and SEQID NO.37 composition, for S4; The sequence of the SEQ ID NO.27 of R32 and SEQ ID NO.38 composition, for S4; The sequence of the SEQ ID NO.28 of R34 and SEQ ID NO.39 composition, for SL13; The sequence of the SEQ ID NO.29 of R32 and SEQ ID NO.40 composition, for SL4; The sequence of the SEQ ID NO.30 of R34 and SEQ ID NO.41 composition, for E10; The sequence of the SEQID NO.31 of R34 and SEQ ID NO.42 composition, for L16; The sequence of the SEQ ID NO.32 of R35 and SEQ ID NO.43 composition and/or for T5; The sequence of the SEQ ID NO.33 of R35 and SEQ ID NO.44 composition.

7. according to the liquid-phase chip described in claim 5 or 6, it is characterized in that, described spacerarm is 5-10 T.

8. for detection of an Auele Specific Primer for ROS1 fusion gene, described Auele Specific Primer is: for C6; The SEQID NO.34 of R32, for C6; The SEQ ID NO.35 of R34, for C5; The SEQ ID NO.36 of R34, for S2; The SEQ ID NO.37 of R32, for S4; The SEQ ID NO.38 of R32, for S4; The SEQ ID NO.39 of R34, for SL13; The SEQ ID NO.40 of R32, for SL4; The SEQ ID NO.41 of R34, for E10; The SEQ ID NO.42 of R34, for L16; The SEQ ID NO.43 of R35 and/or for T5; The SEQ ID NO.44 of R35.