CN103805686B

CN103805686B - PCR primer, kit and liquid-phase chip that ROS1 fusion detects

Info

Publication number: CN103805686B
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: 2016-05-18
Anticipated expiration: 2032-11-09
Also published as: CN103805686A

Abstract

The invention discloses the PCR primer, kit and the liquid-phase chip that detect ROS1 fusion. Described liquid-phase chip comprises: pcr amplification primer; The ASPE primer being formed by tag sequence and Auele Specific Primer, described specific primer sequence is: for C6; The SEQ of R32 ID NO.34, for C6; The SEQ of R34 ID NO.35, for C5; The SEQ of R34 ID NO.36, for S2; The SEQ of R32 ID NO.37, for S4; The SEQ of R32 ID NO.38, for S4; The SEQ of R34 ID NO.39, for SL13; The SEQ of R32 ID NO.40, for SL4; The SEQ of R34 ID NO.41, for E10; The SEQ of R34 ID NO.42, for L16; The SEQ of R35 ID NO.43 and/or for T5; The SEQ of R35 ID NO.44; Microballoon. Liquid-phase chip of the present invention has extraordinary signal-noise ratio, can a step complete 11 kinds of amplifications of merging hypotype, and Auele Specific Primer has extraordinary specificity.

Description

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

Technical field

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

Technical background

V-rosUR2 sarcoma virus oncogene homologue 1(v-rosavianUR2sarcomaviraloncogeneHomolog1, ROS1), within 1987, RabinM. clones and v-ros sarcoma growth related gene, and called after ROS gene.ROS1 gene is the 32nd, and 34 and 35 extron places exist breakpoint, can merge with several genes. ROS1 fusion quiltTurn out to be new lung cancer and drive gene, in the formation of adenocarcinoma of lung, bringing into play significant role. Hold in June, 2012 the 48thIn the annual meeting of American Society of Clinical Oncology (ASCO), the Shaw of masschusetts, u.s.a hospital general reported first about lung cancer recruitTarget spot ROS1 gene fusion patient's I clinical trial phase, clinical testing 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 with regard to ROS1 fusionResearch.

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

CD74 is MHC-II quasi-molecule associated constant chain (majorhistocompatibilitycomplexII-Associatedinvariantchain, Ii), be mainly expressed in the antigens such as BMDC, mononuclear macrophage, B cell and carryBeing cell (antigenpresentingcells, APC), is to be connected to one with new synthetic MHC-II quasi-molecule in endoplasmic reticulumWork the accessory molecule that forms nine aggressiveness, belong to II type membrane molecule, relevant with antigen presentation function. Research is discovery also, and CD74 molecule alsoAt endothelial cell, tumor cells expression, with the generation of corresponding disease, develop relevant. Vascular endothelial cell is to be positioned at blood vesselCell monolayer, there is secretion vaso-active substance, participate in inflammatory cell and immunocyte migration, maintain angiokinesis etc. in many waysFace biological function. At present research shows, the 6th extron of CD74 gene easily and ROS1 gene merge, this fusionThe incidence detecting is 1%. CD74-ROS1 fusion has sensitiveness to gram azoles for Buddhist nun (crizotinib) medicine.

2) SDC4-ROS1 fusion

Syndecan-4(Syndecan-4 is called for short SDC4) be by Heparan sulfate and chondroitin sulfate chain groupBecome, belong to transmembrane protein glycan family, can be combined and serve as coreceptor with multiple heparin class growth factor, conditioning signal transduction,Meanwhile, SDC4 is as cyto-architectural constituent, also participate in sticking between iuntercellular, cell and extracellular matrix, grow because ofThe behaviors such as sub-combination and activation, macromolecular substances transhipment, immune response. The facilitation that SDC4 forms keloid, in scarIn trace pimple, express higher than normal skin tissue, this high expressed promotes fibroblastic propagation in keloid. Grind at presentStudy carefully and show, SDC4 gene the 2nd, 4 extrons easily and ROS1 gene merge.

3) SLC34A2-ROS1 fusion

Solute Transport protein family 34 member 2(solutecarrierfamily34member2, SLC34A2) geneBe positioned on 4p15.31-p15.2. SLC34A2 belongs to one of member of Solute Transport protein family SLC34, strongly expressed in lung,The Pi transport protein NaPi-IIb that coding Na relies on plays an important role in the balance that maintains Phos in body. HighConcentration dexamethasone promotes the SLC34A2 gene expression of people's alveolar epithelial cells and calcium, the phosphorus transporter of extracellular fluid. At present, existIn Lines sample, detect SLC34A2-ROS1 fusion. Research is found, SLC34A2 baseBecause the 4th, 13 extrons are easy and ROS1 gene merges.

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

One of Ezrin albumen is ERM(Ezrin-Radixin-Moesin) family member, mainly participate in cytoskeleton and born of the same parentsConnection between film. Studies have shown that, ezrin gene is many in the cancer of the esophagus, cancer of pancreas, carcinoma of endometrium, prostate cancer and otherPlant in tumour cell and cross and express. Be rich in leucine repetitive sequence immunoglobulin-like albumen 3(leucine-richrepeatsAndimmunoglobin-likedomains3, LRIG3) be one of LRIG gene family member, in kinds of tumors, all haveExpress and lower. Studies have shown that, LRIG3 gene overexpression can reduce invasion and attack and the transfer ability of glioma cell, accelerates cell and withersDie, suppress generation and the development of glioma. Tropomyosin 3 genes (tropomyosin3, TPM3) are in contraction of muscle processOne of important albumen regulatory gene, it exists with actin double helix is parallel in muscle fibre filament, can affect and regulate and controlInteraction between actomyosin. TPM3 gene mutation 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 ROS1Gene the 34th extron merges, and LRIG3 gene the 16th extron and ROS1 gene the 35th extron merge, outside TPM3 gene the 5thAobvious son merges with ROS1 gene the 35th extron.

At present, hybridize for most of RT-PCR method and the on-radiation coordination of adopting of research of RET fusionFISH method detects. RT-PCR method is to draw for type and the amalgamation mode design of known RET fusionThing, obtains RNA reverse transcription after cDNA, detects by the mode of pcr amplification object fragment, 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, testsJourney is too loaded down with trivial details, and the reagent type needing is various, waste time and energy, and whether it can only detect fusion and exist, can not be accurateDraw concrete fused type, sensitivity 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, and described PCR primer can be used forSeparately or the C6 of 11 kinds of gene fusion hypotype: CD74-ROS1 of parallel detection ROS1 fusion; 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, for C6; The SEQIDNO.1 of R32 and SEQIDNO.5, pinTo C6; R34, C5; The SEQIDNO.2 of R34 and SEQIDNO.5, for S2; R32, S4; The SEQIDNO.1 of R32 andSEQIDNO.6, for S4; The SEQIDNO.2 of R34 and SEQIDNO.6, for SL13; The SEQIDNO.1 of R32 andSEQIDNO.7, for SL4; The SEQIDNO.3 of R34 and SEQIDNO.8, for E10; The SEQIDNO.4 of R34 andSEQIDNO.9, for L16; The SEQIDNO.4 of R35 and SEQIDNO.10 and/or for T5; The SEQID of R35NO.4 and SEQIDNO.11.

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

The technical scheme that realizes this object is as follows:

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

In an embodiment, described PCR kit also includes positive reference substance therein, and described positive reference substance is:For C6; R32's contains SEQIDNO.1 and the nucleic acid fragment with the sequence of SEQIDNO.5 reverse complemental pairing, forC6; R34, C5; R34's contains SEQIDNO.2 and nucleic acid fragment, pin with the sequence of SEQIDNO.5 reverse complemental pairingTo S2; R32, S4; R32 contain SEQIDNO.1 and with the nucleic acid fragment of the sequence of SEQIDNO.6 reverse complemental pairing,For S4; R34 contain SEQIDNO.2 and with the nucleic acid fragment of the sequence of SEQIDNO.6 reverse complemental pairing, forSL13; R32 contain SEQIDNO.1 and with the nucleic acid fragment of the sequence of SEQIDNO.7 reverse complemental pairing, for SL4;R34 contain SEQIDNO.3 and with the nucleic acid fragment of the sequence of SEQIDNO.8 reverse complemental pairing, for E10; R34'sContain SEQIDNO.4 and with the nucleic acid fragment of the sequence of SEQIDNO.9 reverse complemental pairing, for L16; R35's containsSEQIDNO.4 and with the nucleic acid fragment of the sequence of SEQIDNO.10 reverse complemental pairing and/or for T5; R35's containsSEQIDNO.4 and with the nucleic acid fragment of the sequence of SEQIDNO.11 reverse complemental pairing.

More preferably, described positive reference substance is: for C6; The SEQIDNO.12 of R32, for C6; The SEQ of R34IDNO.13, for C5; The SEQIDNO.14 of R34, for S2; The SEQIDNO.15 of R32, for S4; The SEQ of R32IDNO.16, for S4; The SEQIDNO.17 of R34, for SL13; The SEQIDNO.18 of R32, for SL4; The SEQ of R34IDNO.19, for E10; The SEQIDNO.20 of R34, for L16; The SEQIDNO.21 of R35 and/or for T5; R35'sSEQIDNO.22。

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

The technical scheme that realizes this object is as follows:

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

(A) above-mentioned PCR primer;

(B). the ASPE primer designing respectively for the different fused type of ROS1 fusion: every ASPE primer is by 5 ' endTag sequence and 3 ' end for the specific primer sequence composition of object fused type, described specific primer sequence is: forC6; The SEQIDNO.34 of R32, for C6; The SEQIDNO.35 of R34, for C5; The SEQIDNO.36 of R34, forS2; The SEQIDNO.37 of R32, for S4; The SEQIDNO.38 of R32, for S4; The SEQIDNO.39 of R34, forSL13; The SEQIDNO.40 of R32, for SL4; The SEQIDNO.41 of R34, for E10; The SEQIDNO.42 of R34, pinTo L16; The SEQIDNO.43 of R35 and/or for T5; The SEQIDNO.44 of R35; Described tag sequence is selected from SEQIDNO.23—SEQIDNO.33；

(C). there is microballoon that anti-tag sequence is coated with, that there is different colours coding, described anti-tag sequence and micro-Ball is also provided with spacerarm sequence in the middle of connecting; Described anti-tag sequence is selected from SEQIDNO.45-SEQIDNO.55, andDescribed 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.

Concrete technical scheme is as follows:

For detection of an Auele Specific Primer for ROS1 fusion, described Auele Specific Primer is: for C6; R32'sSEQIDNO.34, for C6; The SEQIDNO.35 of R34, for C5; The SEQIDNO.36 of R34, for S2; The SEQ of R32IDNO.37, for S4; The SEQIDNO.38 of R32, for S4; The SEQIDNO.39 of R34, for SL13; The SEQ of R32IDNO.40, for SL4; The SEQIDNO.41 of R34, for E10; The SEQIDNO.42 of R34, for L16; The SEQ of R35IDNO.43 and/or for T5; The SEQIDNO.44 of R35.

Major advantage of the present invention is:

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

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

3, it is right as the positive that detection kit of the present invention is used the plasmid vector that contains target detection fusion hypotype sequenceAccording to product, the accuracy and the reliability that detect are further ensured.

4, the liquid-phase chip of the prepared ROS1 fusion of the present invention has extraordinary signal-noise ratio, designedProbe and anti-tag sequence between substantially there is not cross reaction, meanwhile, the ASPE type specificity of the present invention designPrimer has extraordinary specificity, can accurately distinguish the fusion hypotype of various types.

5, the needed time of detection method provided by the present invention, well below conventional sequencing technologies, meets reality especiallyThe needs of border application.

6, not only to have overcome traditional solid phase chip sensitiveness not high in the present invention, the poor defect of repeatability of testing result,Existing liquid-phase chip technology is improved simultaneously, made prepared microballoon can be applicable to different test items, have veryStrong expansion. The fluorescence signal value detecting improves greatly, thereby the sensitivity detecting is further enhanced, signal to noise ratioStrengthen, testing 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.

Detailed description of the invention

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

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

One, RT-PCR amplification

1, reverse transcription

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

2, pcr amplification

Fused type in table 1 is 11 kinds of ROS1 fusion hypotypes of target detection of the present invention, according to this fusionThe nucleic acid composition characteristic of sequence, utilizes Primer5.0 design forward primer and reverse primer. Use the cDNA of step 1 gained to doFor template, to the amplification that walks abreast of 11 kinds of target detection fusion hypotypes.

All primers are synthesized by Invitrogen company. Every primer after synthetic is used respectively 10mmol/LTrisBuffer is mixed with the storage liquid of 300pmol/mL.

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

Table 1ROS1 fusion hypotype amplimer

Two, positive reference substance

The present invention is according to the fusion sequence feature of 11 kinds of hypotypes of target detection ROS1 fusion, and designing nucleic acid fragment is doneFor the positive reference substance detecting, this positive reference substance is the forward primer that contains amplimer in table 1 and reverse with reverse primerThe nucleic acid fragment of the sequence of complementary pairing, the nucleic acid fragment between its forward primer and the sequence of reverse primer reverse complemental pairingCan be human genomic sequence, can be also the nucleotide sequence that derives from the inhuman source such as plant, microorganism, and the sun of synthesizedProperty the clip size of reference substance and table 1 in the amplified production of corresponding target detection fused type in the same size, exist thereby realizeThe effect of positive control in amplification procedure, meanwhile, (for example: when use derives from plant, microorganism realizes other experiment purposeWhen the nucleotide sequence in inhuman source, can avoid human genomic sequence in positive reference substance to detect the positive dirt of sample to otherDye etc. ) in detection method of the present invention, detect fusion hypotype for all types of target, adopt and insert corresponding human genome orderRow are as positive reference substance. Specifically, in the SEQIDNO.12 ~ SEQIDNO.22 in table 2, trust money is intelligence biotechnology (north onlyCapital) Co., Ltd carries out the synthetic of target insetion sequence, purified composition sequence is inserted on PMD18-T carrier, soAfter be converted in bacillus coli DH 5 ɑ competent cell, screen by blue hickie, 11 kinds of ROS1 that establishing target detects merge basesBecause hypotype recombinant plasmid dna is as positive reference substance. 11 kinds of recombinant plasmids that build are correct through two-way DNA sequencing qualification. ExtractPlasmid, ultraviolet specrophotometer is quantitative, is diluted to 5.0 × 10¹⁰Copies/ml is preserved in-20 ° of C.

The insetion sequence of table 2ROS1 fusion hypotype positive control plasmid

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

1, pcr amplification

Utilize the pcr amplification primer described in table 1 in embodiment 1, to mRNA reverse transcription product and the sun of target detection sampleProperty control plasmid mixed liquor carry out pcr amplification, meanwhile, for the ease of using conventional electrophoretic techniques to distinguish pcr amplification band, according toThe size (as shown in table 1) of PCR product, is divided into 2 by 11 kinds of fused type and the corresponding positive control plasmid thereof of target detectionGroup, carries out respectively 2 independently pcr amplifications simultaneously, and uses respectively Ago-Gel and polyacrylamide gel to detect,According to the size of amplified production, PCR primer is divided into required classification, and detects by electrophoretic techniques, belong to art technologyConventional method. This 2 group reaction system is respectively:

First prepare PCR primer working solution: according to 2 PCR groups in above table, respectively get respectively in table 1 correspondingPrimer storage liquid 100ul in 1.5ml microcentrifugal tube, mix, be 2 groups of multiple PCR primer working solutions.

In like manner, preparation positive control plasmid working solution, according to the type of fusion in above table, will be corresponding positiveControl plasmid is put in same 1.5ml microcentrifugal tube, mixes, and forms positive control plasmid working solution.

2 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 CSave 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 target detectionThe mRNA reverse transcription product of sample, if there is the hypotype of certain fused type, amplifies respective strap, if do not contain in sampleThe hypotype of certain fused type, can not amplify corresponding band, and this amplified production can pass through Ago-Gel, polyacrylamideThe conventional method of the art technology such as amine gel, Southern hybridization, solid phase chip, liquid-phase chip detects, thereby determines whetherThere is certain fusion hypotype. Refer to Fig. 1 to Fig. 2, Fig. 1-Fig. 2 is respectively polyacrylamide gel and detects amplified productionElectrophoresis picture.

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

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

One, ASPE primer

For the C6 of the CD74-ROS1 of target detection ROS1 fusion; R32, C6; R34, C5; R34; SDC4-ROS1'sS2; R32, S4; R32, S4; R34; The SL13 of SLC34A2-ROS1; R32, SL4; R34; The E10 of EZR-ROS1; R34; LRIG3-The L16 of ROS1; The T5 of R35 and TPM3-ROS1; R35 is the specific primer sequence of totally 11 kinds of fusion hypotypes design. ASPE primer by" Tag+ specific primer sequence " composition. 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 for anti-tag sequence on corresponding microballoonSequence, 3 ' end is saltant type or wild type specific primer sequence (as shown in Table 3 above). All ASPE primers byInvitrogen company is synthetic. Every primer after synthetic is mixed with 100pmol/mL with 10mmol/LTrisBuffer respectivelyStorage liquid.

Two, the coated microballoon of anti-tag sequence

According to designed ASPE specific primer sequence, select tag sequence, reduce to greatest extent each microballoonBetween anti-tag sequence and tag and the ASPE specific primer sequence secondary structure that may form, 11 kinds of microballoons of selectionOn numbering 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. Anti-tagBetween sequence and microballoon, be connected with the spacerarm order of 5-10 T, before each anti-tag sequence, add the preceding paragraph 5-10 TSpacerarm sequence, anti-tag sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd. By synthetic anti-Tag sequence sterilizing ddH₂O is made into the storage liquid of 100nmol/ml. The coated process of microballoon is as follows:

Get respectively 5 × 10⁶The microballoon (purchased from Luminex company) of the carboxylated of individual above-mentioned numbering is suspended in 50ulIn the MES solution of 0.1mol/L (pH4.5), add the synthetic anti-tag molecule (100nmol/ml) of 10ul. Preparation 10ng/mlEDC(N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide) (purchased from PierceChemical company)Working solution. Toward the EDC working solution that adds 2.5ul in microballoon suspension, constant-temperature incubation 30 minutes, then add the EDC work of 2.5ulLiquid, then constant-temperature incubation 30 minutes. After reaction finishes, the Tween-20 with 0.02% washs once, then washes with 0.1% SDS liquidWash once. The microballoon that is coated with anti-tag sequence after washing is resuspended in to the Tris-EDTA solution [10mmol/L of 100ulTris(pH8.0),, in 1mmol/LEDTA, 2-8 DEG C 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 examination in embodiment 1Agent box carries out the parallel amplification of PCR to 11 of target detection kinds of fusion hypotypes and positive reference substance, concrete forward primer andReverse primer, as shown in table 1, concrete operating procedure is shown in embodiment 2, uses this embodiment, can realize 11 kinds of fusionsThe parallel detection of hypotype.

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

The formula of described various solution is as follows:

MES buffer solution (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 DEG C.

ExoSAP-IT 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 sampleMRNA carries out reverse transcription, 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, to the mRNA of target detection sampleReverse transcription product and positive reference substance carry out pcr amplification, realize the parallel amplification of the target sequence of 11 kinds of fusion hypotypes, oneStep amplifies 11 target sequences that contain fusion hypotype, and product size is as shown in table 1. Primer sequence (SEQIDNO.1~ SEQIDNO.11) see shown in above-mentioned table 1.

First prepare PCR primer working solution: the primer storage liquid of respectively getting respectively SEQIDNO.1 ~ SEQIDNO.11100ul, in 1.5ml microcentrifugal tube, mixes and is multiple PCR primer working solution. Multi-PRC reaction system is as follows:

Three, the enzyme of PCR product is cut processing

With reference to the explanation of ExoSAP-IT 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. Enzyme is cut product after treatment and is directly used inFollow-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 course of reaction, mix biotin markThe dCTP of note, thus make multiple biotin labeling on reacted product band.

First the ASPE primer working solution that preparation mixes: get the each 10ul of corresponding ASPE primer storage liquid in 1.5ml traceIn centrifuge tube, add 10mmol/LTrisBuffer to mend to 200ul, mix and be ASPE mix primer working solution.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 reaction

1. according to the ASPE primer of design, select 11 kinds of microballoons that are coated with anti-tag sequence as described in Example 3(microballoon concentration is 2.5 × 10⁵Individual/ml).

2. get respectively the microballoon of every kind of numbering of 1ul in the microcentrifugal 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 25ul₂O；

6. get the ASPE reactant liquor of 5-25ul in corresponding hole, use ddH₂O complements to 50ul;

7. encase 96 orifice plates with lucifuge with masking foil, 95 DEG C of 60s, 37 DEG C 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 strepto-parent of 10ug/mlAnd element-phycoerythrin (SA-PE);

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

Six, result detects and data analysis

Reaction afterproduct detects by Luminex serial analysis instrument. Taking saltant type fluorescent value (MFI) be greater than 100 asCut-off value, in the time that the MFI value of saltant type detection is greater than 100, judges that this sample exists this mutation type, otherwise judges this sampleThis is corresponding wild type.

Use this method to detect the ROS1 fusion hypotype of great amount of samples, detect with liquid-phase chip result and do with PCR sequencing PCRContrast detects the RT-PCR product in embodiment 1 simultaneously, calculates coincideing of method testing result provided by the present inventionRate. This method detects 20 increments ROS1 fusion hypotype testing result and the sequencing result rate of coincideing originally and reaches 100%. VisibleROS1 fusion hypotype provided by the present invention detects liquid-phase chip can detect 11 kinds of ROS1 fusion Asias exactlyType, 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 12 -->
						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 testing result	Order-checking testing 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; R32, S2; R32; SL13; R32; E10; R34; L16; R35 and T5; R35 hypotypeDetection liquid-phase chip is example, and the specific primer sequence of holding for these 6 kinds of fusion hypotype design ASPE primers 3 ' is (as table 3Shown in), the Tag sequence of ASPE primer 5 ' end is selected from SEQIDNO.23-SEQIDNO.33, corresponding, is coated in micro-The anti-tag sequence with corresponding tag sequence complementary pairing on ball is selected from SEQIDNO.45-SEQIDNO.55. Specifically establishMeter is as shown in following table (table 8). Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. strictly according to the factsExecute described in example 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, carries out sample 31-50 by testing process described in embodiment 2 and methodDetect, testing 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 hypotype Analysis of test results

Catalogue number(Cat.No.)	Liquid-phase chip detects knot	Order-checking testing 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 tagSequence, its result is still reliable and stable, and concrete data are omitted. And ASPE primer selects tag sequence and specificity in embodiment 3 to drawWhen the collocation of thing sequence, effect better (signal to noise ratio is better), referring to the present embodiment test group 1. Other different tag sequences and specificityPrimer 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 parallel detection liquid-phase chip of the present invention, carry out the detection of cross reacting rate. Get positive control matterGrain, the C6 of detection 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; R35 and TPM3-The T5 of ROS1; R35 is the cross reacting rate of totally 11 kinds of fusion hypotypes.

Two, operating procedure

1, the mixed liquor of preparation positive control plasmid (being called for short: sample plasmid), the concentration that this mixed liquor contains every kind of plasmidBe 10⁶Copy/uL, loading 10uL, repeats 3 times.

2, the mixed liquor of positive control plasmid is carried out to pcr amplification, a step amplifies 11 and contains fusion hypotypeTarget sequence, product size is as shown in table 1.

Preparation PCR primer working solution: the primer storage liquid 100ul that respectively gets respectively SEQIDNO.1 ~ SEQIDNO.11 inIn 1.5ml microcentrifugal tube, mix and be multiple PCR primer working solution. 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 solution: get correspondingThe each 10ul of ASPE primer storage liquid, in 1.5ml microcentrifugal tube, adds 10mmol/LTrisBuffer to mend to 200ul, mixedClose and be evenly ASPE primer working solution. Prepare respectively 11 kinds of ASPE working solutions for different fused type, and below usingSystem, carries out respectively ASPE extension. The system of ASPE reaction is as follows:

Five, hybridization reaction (as described in Example 4)

Six, result detects and data analysis

Reaction afterproduct detects by Luminex serial analysis instrument.

Table 12 sample plasmid testing result

The selection of embodiment 7ROS1 fusion 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 C6; R34; S4; R32; SL13; The detection liquid-phase chip of R32 hypotype is example, with this sudden change positionThe complementary series forward or backwards of some place target sequence is template, respectively for C6; R34; S4; R32; SL13; R32 hypotype is establishedThe specific primer sequence of meter ASPE primer 3 ' end, comprise in the embodiment of the present invention 1 preferred specific primer sequence and 2 standbyThe specific primer sequence of choosing, as shown in table 13.

Table 13 specific primer sequence

With ROS1 fusion 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 fixingFor the optimum efficiency sequence in embodiment 1, and select the anti-tag sequence of answering in contrast, specific design is as following table (table 14)Shown in. Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. as embodiment 3 and embodimentDescribed in 4.

Design prepared by table 14 liquid-phase chip

Two, sample detection

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

Table 15 pattern detection result (MFI)

Table 16 sample ROS1 fusion hypotype Analysis of test results

Catalogue number(Cat.No.)	Liquid-phase chip detects knot	Order-checking testing 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, effectFruit better (signal to noise ratio is better), referring to the present embodiment test group 4. Other derive from place, target detection site sequence forward orThe different specific primer sequences of reverse complementary sequence and the collocation of tag 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 economizedSlightly.

The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but alsoCan not therefore be interpreted as the restriction to the scope of the claims of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to guarantor of the present inventionProtect scope. 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, is characterized in that, described PCR primer is for C6; The SEQID of R32NO.1 and SEQIDNO.5, and be selected from for C6; R34, C5; The SEQIDNO.2 of R34 and SEQIDNO.5, for S2;R32, S4; The SEQIDNO.1 of R32 and SEQIDNO.6, for S4; The SEQIDNO.2 of R34 and SEQIDNO.6, pinTo SL13; The SEQIDNO.1 of R32 and SEQIDNO.7, for SL4; The SEQIDNO.3 of R34 and SEQIDNO.8, pinTo E10; The SEQIDNO.4 of R34 and SEQIDNO.9, for L16; The SEQIDNO.4 of R35 and SEQIDNO.10 andFor T5; In the SEQIDNO.4 of R35 and SEQIDNO.11 at least one group.

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

3. PCR kit according to claim 2, is characterized in that, described positive reference substance is: for C6; The SEQ of R32IDNO.12, for C6; The SEQIDNO.13 of R34, for C5; The SEQIDNO.14 of R34, for S2; The SEQID of R32NO.15, for S4; The SEQIDNO.16 of R32, for S4; The SEQIDNO.17 of R34, for SL13; The SEQID of R32NO.18, for SL4; The SEQIDNO.19 of R34, for E10; The SEQIDNO.20 of R34, for L16; The SEQID of R35NO.21 and/or for T5; The SEQIDNO.22 of R35.

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

(A) PCR primer claimed in claim 1;

(B). the ASPE primer designing respectively for the different fused type of ROS1 fusion: every ASPE primer is by 5 ' endTag sequence and 3 ' end are for the specific primer sequence composition of object fused type, and described specific primer sequence is: forC6; The SEQIDNO.34 of R32, and be selected from for C6; The SEQIDNO.35 of R34, for C5; The SEQID of R34NO.36, for S2; The SEQIDNO.37 of R32, for S4; The SEQIDNO.38 of R32, for S4; The SEQID of R34NO.39, for SL13; The SEQIDNO.40 of R32, for SL4; The SEQIDNO.41 of R34, for E10; The SEQ of R34IDNO.42, for L16; The SEQIDNO.43 of R35 and for T5; At least one in the SEQIDNO.44 of R35; InstituteState tag sequence and be selected from SEQIDNO.23-SEQIDNO.33;

(C). have microballoon that anti-tag sequence is coated with, that have different colours coding, described anti-tag sequence and microballoon connectIn the middle of connecing, be also provided with spacerarm sequence; Described anti-tag sequence is selected from SEQIDNO.45-SEQIDNO.55, and described inAnti-tag sequence can be correspondingly with (B) in selected tag sequence complementary pairing.

5. require the liquid-phase chip described in 4 according to power, it is characterized in that, described ASPE primer is: for C6; R32 by SEQIDThe sequence of NO.23 and SEQIDNO.34 composition, and be selected from for C6; The SEQIDNO.24 of R34 and SEQIDNO.35Composition sequence, for C5; The sequence of the SEQIDNO.25 of R34 and SEQIDNO.36 composition, for S2; The SEQ of R32The sequence of IDNO.26 and SEQIDNO.37 composition, for S4; The SEQIDNO.27 of R32 and SEQIDNO.38 compositionSequence, for S4; The sequence of the SEQIDNO.28 of R34 and SEQIDNO.39 composition, for SL13; The SEQID of R32The sequence of NO.29 and SEQIDNO.40 composition, for SL4; The order of the SEQIDNO.30 of R34 and SEQIDNO.41 compositionRow, for E10; The sequence of the SEQIDNO.31 of R34 and SEQIDNO.42 composition, for L16; The SEQID of R35The sequence of NO.32 and SEQIDNO.43 composition and for T5; The SEQIDNO.33 of R35 and SEQIDNO.44 compositionAt least one in sequence.

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