CN103805688B - The PCR primer that RET fusion gene detects, test kit and liquid-phase chip - Google Patents
The PCR primer that RET fusion gene detects, test kit and liquid-phase chip Download PDFInfo
- Publication number
- CN103805688B CN103805688B CN201210448645.2A CN201210448645A CN103805688B CN 103805688 B CN103805688 B CN 103805688B CN 201210448645 A CN201210448645 A CN 201210448645A CN 103805688 B CN103805688 B CN 103805688B
- Authority
- CN
- China
- Prior art keywords
- seqidno
- sequence
- type
- primer
- wild
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the PCR primer, test kit and the liquid-phase chip that detect RET fusion gene.Described liquid-phase chip comprises: pcr amplification primer; The ASPE primer be made up of tag sequence and Auele Specific Primer, described specific primer sequence is: for K15; The SEQ of R12 ID NO.23, for K16; The SEQ of R12 ID NO.24, for K22; The SEQ of R12 ID NO.25, for K23; The SEQ of R12 ID NO.26, for K24; The SEQ of R8 ID NO.27, for K24; The SEQ of R11 ID NO.28 and/or for C1; The SEQ of R12 ID NO.29; Microballoon.Liquid-phase chip of the present invention has extraordinary signal-noise ratio, a step can complete the amplification that 7 kinds merge hypotype, and Auele Specific Primer has extraordinary specificity.
Description
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 RET 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).In Mammals, gene fusion is one of reason causing some disease (as cancer) to occur.Gene fusion phenomenon is found in multiple blood and soft-tissue cancers the earliest, as leukemia, lymphoma etc.RET proto-oncogene is positioned on euchromosome 11q11.2, is about 60000bp, at least containing 20 exons, and encoding tyrosine kinases acceptor.Current research shows being related closely of the disease such as RET transgenation and fMTC, sMTC, multiple endocrine tumors 2A and 2B type, congenital megacolon.Research finds further, and the morbidity of RET fusion gene and lung cancer, thyroid carcinoma is closely related.Investigator finds that from 24 routine patients with lung cancer 1 example has KIF5B-RET to merge variation.Investigator finds 1 example (0.8%) further, in 405 example Japan and Korea S's lung cancer sample, finds that 9 examples (2%) patient exists KIF5B-RET gene fusion in 121 routine European lung cancer samples.Detect in the 667 routine lung cancer samples analyzed all, KIF5B-RET gene fusion frequency is about 1.8%(12/667 example), and in the lung cancer not having EGFR, KRAS, HER2, ALK, ROS1 to make a variation, the fusion frequency of RET gene is up to 6.3%(10/159 example).It should be noted that, the new fusion gene that this finds in NSCLC for KIF5B-RET, the Ba/F3 cell of transfection expression KIF5B-RET fusion gene shows the high expression level of RET and phosphorylated-activated, and experiment in vitro finds, Mutiple Targets medicine such as Xarelto, Sutent and ZD6474 (vandetanib) all can suppress the propagation of this cell.
CCDC6-RET is newfound fusion gene in non-smoking patients with lung adenocarcinoma, and information biology and experiment results show, there is a new lung cancer Disease-causing gene and merge CCDC6-RET in about tumor sample of 10% (2/24).RET is a receptor tyrosine kinase, expresses lower in normal lung tissue; And the extracellular domain of CCDC6 and the kinase activity district of RET have been merged in this transgenation, often express higher in lung cancer sample, and this may be the key point causing lung cancer morbidity.
At present, the research major part for RET fusion gene adopts RT-PCR method and on-radiation coordination hybridization FISH method to detect.RT-PCR method is to design primer for the type of known RET fusion gene and amalgamation mode, after RNA reverse transcription is obtained cDNA, detected by the mode of pcr amplification object fragment, then there is sensitivity low, sample easily pollutes, shortcoming that false positive rate is high.Although and nonradioactivein situhybridization technology FISH method is comparatively directly perceived, but process of the test is too loaded down with trivial details, the reagent type needed 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, thus limits the application of this method to a certain extent.
Summary of the invention
An object of the present invention is to provide a kind of PCR primer detecting RET fusion gene, and described PCR primer can be used for separately or 7 kinds of gene fusion hypotypes of parallel detection RET fusion gene: the K15 of KIF5B-RET fusion gene; R12, K16; R12, K22; R12, K23; R12, K24; R8, K24; The C1 of R11 and CCDC6-RET fusion gene; R12.
The technical scheme realizing above-mentioned purpose is as follows:
Detect a PCR primer for RET fusion gene, described PCR primer is: for K15; SEQIDNO.1 and SEQIDNO.4 of R12, for K16; SEQIDNO.1 and SEQIDNO.5 of R12, for K22; R12 and K23; SEQIDNO.1 and SEQIDNO.6 of R12, for K24; SEQIDNO.2 and SEQIDNO.7 of R8, for K24; SEQIDNO.3 and SEQIDNO.7 of R11 and/or for C1; SEQIDNO.1 and SEQIDNO.8 of R12.
Another object of the present invention is to provide a kind of PCR kit detecting RET fusion gene.
The technical scheme realizing above-mentioned purpose is as follows:
Detect a PCR kit for RET 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: described positive reference substance is: for K15; The nucleic acid fragment containing SEQIDNO.1 and the sequence of matching with SEQIDNO.4 reverse complemental of R12, for K16; The nucleic acid fragment containing SEQIDNO.1 and the sequence of matching with SEQIDNO.5 reverse complemental of R12, for K22; R12 and K23; The nucleic acid fragment containing SEQIDNO.1 and the sequence of matching with SEQIDNO.6 reverse complemental of R12, for K24; The nucleic acid fragment containing SEQIDNO.2 and the sequence of matching with SEQIDNO.7 reverse complemental of R8, for K24; R11 containing SEQIDNO.3 and the nucleic acid fragment of sequence matched with SEQIDNO.7 reverse complemental and/or for C1; R12 containing SEQIDNO.1 and the nucleic acid fragment of sequence that matches with SEQIDNO.8 reverse complemental.
More preferably, described positive reference substance is: for K15; The SEQIDNO.9 of R12, for K16; The SEQIDNO.10 of R12, for K22; The SEQIDNO.11 of R12, for K23; The SEQIDNO.12 of R12, for K24; The SEQIDNO.13 of R8, for K24; The SEQIDNO.14 of R11 and/or for C1; The SEQIDNO.15 of R12.
Another object of the present invention is to provide a kind of liquid-phase chip detecting RET fusion gene and detect.The liquid-phase chip that described RET fusion gene detects can be used for separately or 7 kinds of gene fusion hypotypes of parallel detection RET fusion gene: the K15 of KIF5B-RET fusion gene; R12, K16; R12, K22; R12, K23; R12, K24; R8, K24; The C1 of R11 and CCDC6-RET fusion gene; R12.
The technical scheme realizing above-mentioned purpose is as follows:
Detect a liquid-phase chip for RET fusion gene, include:
(A) above-mentioned PCR primer;
(B). the ASPE primer for the different fused type of RET 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 K15; The SEQIDNO.23 of R12, for K16; The SEQIDNO.24 of R12, for K22; The SEQIDNO.25 of R12, for K23; The SEQIDNO.26 of R12, for K24; The SEQIDNO.27 of R8, for K24; The SEQIDNO.28 of R11 and/or for C1; The SEQIDNO.29 of R12; Described tag sequence is selected from SEQIDNO.16 ~ SEQIDNO.22;
(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.30 ~ SEQIDNO.36, and described anti-tag sequence can correspondingly be matched with selected tag complementary in (B).
Wherein in an embodiment, described ASPE primer is: for K15; The sequence be made up of SEQIDNO.16 and SEQIDNO.23 of R12, for K16; The sequence be made up of SEQIDNO.17 and SEQIDNO.24 of R12, for K22; The sequence be made up of SEQIDNO.18 and SEQIDNO.25 of R12, for K23; The sequence be made up of SEQIDNO.19 and SEQIDNO.26 of R12, for K24; The sequence be made up of SEQIDNO.20 and SEQIDNO.27 of R8, for K24; The sequence be made up of SEQIDNO.21 and SEQIDNO.28 of R11 and/or for C1; The sequence be made up of SEQIDNO.22 and SEQIDNO.29 of R12.
Another object of the present invention is to provide a kind of Auele Specific Primer for detecting RET fusion gene.
Concrete technical scheme is as follows:
For detecting an Auele Specific Primer for RET fusion gene, described Auele Specific Primer is for K15; The SEQIDNO.23 of R12, for K16; The SEQIDNO.24 of R12, for K22; The SEQIDNO.25 of R12, for K23; The SEQIDNO.26 of R12, for K24; The SEQIDNO.27 of R8, for K24; The SEQIDNO.28 of R11 and/or for C1; The SEQIDNO.29 of R12.
Major advantage of the present invention is:
1, the detected result of RET fusion gene detection liquid phase chip reagent box provided by the present invention and sequencing coincide rate 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 7 kinds of 1RT 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.
3, 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.
The liquid-phase chip of the RET fusion gene 4, 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.
5, the time required for detection method provided by the present invention 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.
Embodiment
Unaccounted normal condition and method in the embodiment of the present invention, the ordinary method adopted 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 production firm provides.
The PCR primer that the fusion gene that embodiment 1 one kinds detects RET fusion gene detects and positive reference substance
One, RT-PCR amplification
1, reverse transcription
For the RET 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 7 kinds of RET 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, parallel amplification is carried out to 7 kinds of target detect fusion gene hypotypes.
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, K15; R12 represents that the exons 15 of KIF5B gene and the exons 12 of RET gene merge.
Table 1RET fusion gene hypotype amplimer
Two, positive reference substance
The present invention is according to the fusion sequence feature of 7 of target detect RET 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 experiment purpose (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 detection method, for various target detect fusion gene hypotype, adopt and insert corresponding human genomic sequence as positive reference substance.Specifically in the SEQIDNO.9 ~ SEQIDNO.15 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, 7 kinds of RET fusion gene hypotype recombinant plasmid dnas that establishing target detects are as positive reference substance.The 7 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 2RET fusion gene hypotype positive control plasmid
Embodiment 2 uses PCR primer in embodiment 1 to detect RET fusion gene
1, pcr amplification
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 7 kinds of fusion gene hypotypes, one step amplifies 7 target sequences containing fusion gene hypotype, product size is as shown in table 1, and amplimer 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:
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, Fig. 1 is 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 7 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 RET fusion gene
One, ASPE primer
For the K15 of target detect RET fusion gene; R12, K16; R12, K22; R12, K23; R12, K24; R8, K24; R11, C1; R12 is totally 7 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 7 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.By the anti-tag sequence sterilizing ddH of synthesis
2o 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 detection method detecting RT-PCR amplified production in embodiment 1, the detection method in embodiment 2 is used to carry out pcr amplification to 7 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.
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 |
| 5MNaCl | 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 2, 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 7 kinds of fusion gene hypotypes, one step amplifies 7 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:
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.
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 7 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 RET fusion gene hypotype of great amount of samples, detect with sequencing and compare with liquid-phase chip result, 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 detect 20 increments this RET fusion gene hypotype detected result and the sequencing result rate of coincideing reach 100%.Visible RET fusion gene hypotype provided by the present invention detects liquid-phase chip can detect 7 kinds of RET fusion gene hypotypes exactly, and result is reliable and stable.
Table 5 pattern detection result (MFI)
Table 6 sample RET analysis of fused genes result
| Catalogue number(Cat.No.) | Liquid-phase chip detects knot | Order-checking detected result |
| 11 | Wild-type | Wild-type |
| 12 | Wild-type | Wild-type |
| 13 | Wild-type | Wild-type |
| 14 | Wild-type | Wild-type |
| 15 | Wild-type | Wild-type |
| 16 | Wild-type | Wild-type |
| 17 | K16; R12 merges | K16; R12 merges |
| 18 | Wild-type | Wild-type |
| 19 | Wild-type | Wild-type |
| 20 | Wild-type | Wild-type |
| 21 | Wild-type | Wild-type |
| 22 | K23; R12 merges | K23; R12 merges |
| 23 | Wild-type | Wild-type |
| 24 | Wild-type | Wild-type |
| 25 | Wild-type | Wild-type |
| 26 | K24; R8 merges | K24; R8 merges |
| 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 K15 of RET fusion gene; R12, K22; R12, K24; The detection liquid-phase chip of R8 type is example, for the specific primer sequence (as shown in table 3) that this 3 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.16-SEQIDNO.22, accordingly, be coated in and microballoon is selected from SEQIDNO.30-SEQIDNO.36 with the anti-tag sequence that corresponding tag complementary matches.Specific design is as shown in following table (table 7).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 7 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:
Table 8 pattern detection result (MFI)
Table 9 sample RET 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 | K23; R12 merges | K23; R12 merges |
| 45 | Wild-type | Wild-type |
| 46 | Wild-type | Wild-type |
| 47 | Wild-type | Wild-type |
| 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 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 4 and the present embodiment, concrete data are omitted.
The cross reacting rate of embodiment 6 liquid-phase chip 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 K15; R12, K16; R12, K22; R12, K23; R12, K24; R8, K24; R11, C1; R12 totally 7 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, repeats 3 times.
2, carry out pcr amplification to the mixed solution of positive control plasmid, a step amplifies 7 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:
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.
Three, the enzyme of PCR primer cuts process (as described in Example 4)
Four, site-specific primer extension reaction (as described in Example 4)
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.
For 7 kinds of gene fusion hypotypes, get corresponding ASPE primer respectively, be mixed with ASPE working fluid: 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 primer working fluid.Prepare 7 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:
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 (as described in Example 4)
Six, result detects and data analysis
Reaction after product is detected by Luminex serial analysis instrument.
Table 10 sample plasmids detection result
The selection of embodiment 7RET 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 RET fusion gene K16; R12; K24; R8; C1; The detection liquid-phase chip of R12 hypotype is example, with the complementary sequence forward or backwards of this place, mutational site target sequence for template, respectively for K16; R12; K24; R8; C1; The specific primer sequence that R12 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, as shown in table 11.
Table 11 specific primer sequence
With RET fusion gene K16; R12; K24; R8; C1; The detection liquid-phase chip of R12 hypotype is example, for K16; R12; K24; R8; C1; R12 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 12).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 12 liquid-phase chip
Two, sample detection
Adopt liquid-phase chip prepared by above-mentioned design, detect sample 51-70 by testing process described in embodiment 2 and method, detected result is as follows:
Table 13 pattern detection result (MFI)
Table 14 sample RET 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 | Wild-type | Wild-type |
| 56 | Wild-type | Wild-type |
| 57 | C1; R12 merges | C1; R12 merges |
| 58 | Wild-type | Wild-type |
| 59 | Wild-type | Wild-type |
| 60 | Wild-type | Wild-type |
| 61 | Wild-type | Wild-type |
| 62 | Wild-type | Wild-type |
| 63 | K16; R12 merges | K16; R12 merges |
| 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, identical with the detected result of the present embodiment with embodiment 4, 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 (6)
1. detect a PCR primer for RET fusion gene, it is characterized in that, described PCR primer is: for K15; SEQIDNO.1 and SEQIDNO.4 of R12, for K16; SEQIDNO.1 and SEQIDNO.5 of R12, for K22; R12 and K23; SEQIDNO.1 and SEQIDNO.6 of R12, for K24; SEQIDNO.2 and SEQIDNO.7 of R8, for K24; SEQIDNO.3 and SEQIDNO.7 of R11 and/or for C1; SEQIDNO.1 and SEQIDNO.8 of R12.
2. detect a PCR kit for RET fusion gene, it is characterized in that, include PCR primer according to claim 1.
3. PCR kit according to claim 2, is characterized in that, also includes positive reference substance, and described positive reference substance is: for K15; The SEQIDNO.9 of R12, for K16; The SEQIDNO.10 of R12, for K22; The SEQIDNO.11 of R12, for K23; The SEQIDNO.12 of R12, for K24; The SEQIDNO.13 of R8, for K24; The SEQIDNO.14 of R11 and/or for C1; The SEQIDNO.15 of R12.
4. a liquid-phase chip for RET fusion gene detection, is characterized in that, include:
(A) PCR primer according to claim 1;
(B). the ASPE primer for the different fused type of RET 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 K15; The SEQIDNO.23 of R12, for K16; The SEQIDNO.24 of R12, for K22; The SEQIDNO.25 of R12, for K23; The SEQIDNO.26 of R12, for K24; The SEQIDNO.27 of R8, for K24; The SEQIDNO.28 of R11 and/or for C1; The SEQIDNO.29 of R12; Described tag sequence is selected from SEQIDNO.16 ~ SEQIDNO.22;
(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.30 ~ SEQIDNO.36, and described anti-tag sequence can correspondingly be matched with selected tag complementary in (B).
5. liquid-phase chip according to claim 4, is characterized in that, described ASPE primer is: for K15; The sequence be made up of SEQIDNO.16 and SEQIDNO.23 of R12, for K16; The sequence be made up of SEQIDNO.17 and SEQIDNO.24 of R12, for K22; The sequence be made up of SEQIDNO.18 and SEQIDNO.25 of R12, for K23; The sequence be made up of SEQIDNO.19 and SEQIDNO.26 of R12, for K24; The sequence be made up of SEQIDNO.20 and SEQIDNO.27 of R8, for K24; The sequence be made up of SEQIDNO.21 and SEQIDNO.28 of R11 and/or for C1; The sequence be made up of SEQIDNO.22 and SEQIDNO.29 of R12.
6. the liquid-phase chip according to claim 4 or 5, is characterized in that, described spacerarm is 5-10 T.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210448645.2A CN103805688B (en) | 2012-11-09 | 2012-11-09 | The PCR primer that RET fusion gene detects, test kit and liquid-phase chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210448645.2A CN103805688B (en) | 2012-11-09 | 2012-11-09 | The PCR primer that RET fusion gene detects, test kit and liquid-phase chip |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103805688A CN103805688A (en) | 2014-05-21 |
| CN103805688B true CN103805688B (en) | 2016-04-27 |
Family
ID=50703073
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210448645.2A Active CN103805688B (en) | 2012-11-09 | 2012-11-09 | The PCR primer that RET fusion gene detects, test kit and liquid-phase chip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103805688B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104790042B (en) * | 2015-05-05 | 2017-01-11 | 天津诺禾致源生物信息科技有限公司 | High-throughput sequencing library and building method thereof |
| CN104975080A (en) * | 2015-05-29 | 2015-10-14 | 北京海思特临床检验所有限公司 | Primers and kit for detecting lung cancer KIF5B-RET fusion gene, and usage method thereof |
| WO2017097697A1 (en) * | 2015-12-08 | 2017-06-15 | Boehringer Ingelheim International Gmbh | Method using a ret fusion gene as a biomarker to select non-small cell lung cancer (nsclc) and thyroid cancer patients for a cancer treatment |
| CN107022610B (en) * | 2017-04-01 | 2019-02-05 | 常州桐树生物科技有限公司 | The detection chip and its application of tumour driving gene |
| CN109971861A (en) * | 2019-05-05 | 2019-07-05 | 上海睿璟生物科技有限公司 | CCDC6-RET fusion detection kit |
| CN110205364B (en) * | 2019-06-28 | 2022-11-15 | 北京和合医学诊断技术股份有限公司 | RET gene mutation detection primer group, kit, system and method |
| CN111909987A (en) * | 2020-07-31 | 2020-11-10 | 浙江科途医学科技有限公司 | Reagent composition, kit and detection system for KIF5B-RET fusion gene detection |
| CN115927564B (en) * | 2022-09-29 | 2023-09-12 | 杭州联川基因诊断技术有限公司 | Primer combination, kit and method for detecting gene fusion in biological sample |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955995A (en) * | 2010-06-17 | 2011-01-26 | 江苏迈迪基因生物科技有限公司 | Combined detection method and diagnostic kit of fusion genes related to lymphoma |
| CN102010906A (en) * | 2010-11-09 | 2011-04-13 | 广州益善生物技术有限公司 | Emb B gene mutation detection specific primers and liquid phase chip |
-
2012
- 2012-11-09 CN CN201210448645.2A patent/CN103805688B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955995A (en) * | 2010-06-17 | 2011-01-26 | 江苏迈迪基因生物科技有限公司 | Combined detection method and diagnostic kit of fusion genes related to lymphoma |
| CN102010906A (en) * | 2010-11-09 | 2011-04-13 | 广州益善生物技术有限公司 | Emb B gene mutation detection specific primers and liquid phase chip |
Non-Patent Citations (1)
| Title |
|---|
| RET，ROS1 and ALK fusions in lung cancer;Kengo Takeuchi et al;《Nature Medicine》;20120212;摘要、第379页左栏第1-2段，第380页图2、补充材料中的Table 1、补充材料第2页第6-35行 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103805688A (en) | 2014-05-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103805688B (en) | The PCR primer that RET fusion gene detects, test kit and liquid-phase chip | |
| CN103805684B (en) | The PCR primer that EML4-ALK fusion gene detects, test kit and liquid-phase chip | |
| CN102994617B (en) | HRAS gene mutation detection specificity primer and liquid chip thereof | |
| CN103805687B (en) | PCR primer, test kit and the liquid-phase chip that ALK fusion gene detects | |
| CN102234686B (en) | Liquid chip for detecting KRAS gene mutation | |
| CN103451267B (en) | TERT detection in Gene Mutation Auele Specific Primer and liquid-phase chip | |
| CN103031367B (en) | VHL (Von Hippel Lindau) genetic mutation detection specific primer and liquid phase chip | |
| CN103031369B (en) | CDKN2A gene mutation detection specific primer and liquid chip | |
| CN103865991B (en) | XRCC2 detection in Gene Mutation Auele Specific Primer and liquid-phase chip | |
| CN102191336B (en) | MYC gene single nucleotide polymorphism (SNP) detection specific primer and liquid-phase chip | |
| CN103805686B (en) | PCR primer, kit and liquid-phase chip that ROS1 fusion detects | |
| CN103571919B (en) | Specific detection primers and detection liquid phase chip for HNF1B gene mutation | |
| CN103031368B (en) | FGFR1 gene mutation detection specific primer and liquid chip | |
| CN103031364B (en) | FGFR3 gene mutation detection specific primer and liquid chip | |
| CN102994622B (en) | MET gene mutation detection specificity primer and liquid chip thereof | |
| CN103865985B (en) | BRCA1 detection in Gene Mutation Auele Specific Primer and liquid-phase chip | |
| CN103849676B (en) | CCDC26 detection in Gene Mutation Auele Specific Primer and liquid-phase chip | |
| CN102994618B (en) | AKT3 gene mutation detection specificity primer and liquid chip thereof | |
| CN103865988B (en) | CASP8 detection in Gene Mutation Auele Specific Primer and liquid-phase chip | |
| CN103451269B (en) | PDLIM5 gene mutation detection specific primer and liquid phase chip | |
| CN102994625B (en) | PIK3R1 gene mutation detection specificity primer and liquid chip thereof | |
| CN103374611B (en) | LIG3 (DNA (deoxyribonucleic acid) Ligase III) gene mutation detection specific primers and liquid chip | |
| CN102952866A (en) | Specific primer and liquid chip for detecting polymorphism of DPYD (dihydropyrimidine dehydrogenase) gene | |
| CN102994616B (en) | MEK1 gene mutation detection specificity primer and liquid chip thereof | |
| CN103031371B (en) | CDH1 gene mutation detection specific primer and liquid chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |