CN103805688A - PCR (Polymerase Chain Reaction) primer, kit and liquid chip for detecting RET fusion gene - Google Patents
PCR (Polymerase Chain Reaction) primer, kit and liquid chip for detecting RET fusion gene Download PDFInfo
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- CN103805688A CN103805688A CN201210448645.2A CN201210448645A CN103805688A CN 103805688 A CN103805688 A CN 103805688A CN 201210448645 A CN201210448645 A CN 201210448645A CN 103805688 A CN103805688 A CN 103805688A
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Abstract
The invention discloses a PCR (Polymerase Chain Reaction) primer, a kit and a liquid chip for detecting an RET fusion gene. The liquid chip comprises a PCR amplification primer, an ASPE (Allele Specific Primer Extension) primer which consists of tag sequences and specific primers, and microspheres, wherein the specific primers comprise the following sequences: SEQ ID NO.23 for K15;R12, SEQ ID NO.24 for K16;R12, SEQ ID NO.25 for K22;R12, SEQ ID NO.26 for K23;R12, SEQ ID NO.27 for K24;R8, SEQ ID NO.28 for K24;R11 and/or SEQ ID NO.29 for C1;R12. The liquid chip disclosed by the invention has a quite good signal to noise ratio and can be used for implementing amplification of seven fusion subtypes through one step, and the specific primers have quite good specificity.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete the PCR primer, test kit and the liquid-phase chip that relate to the detection of RET fusion gene.
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 (Gene fusions).In Mammals, gene fusion is one of reason causing some disease (as cancer) generation.Gene fusion phenomenon is found in multiple blood and soft-tissue cancers the earliest, as leukemia, lymphoma etc.It is upper that RET proto-oncogene is positioned at euchromosome 11q11.2, is about 60000bp, at least containing 20 exons, and coding tyrosine kinase receptor.At present research shows that being related of the diseases such as RET transgenation and fMT C, sMT C, multiple endocrine tumors 2A and 2B type, congenital megacolon is close.Research is discovery 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 further finds 1 example (0.8%), in 405 routine 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.In the 667 routine lung cancer samples of analyzing in all detections, KIF5B-RET gene fusion frequency is about 1.8%(12/667 example), and in the lung cancer that does not have 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, this new fusion gene of finding in NSCLC for KIF5B-RET, the Ba/F3 cell of transfection expression KIF5B-RET fusion gene has demonstrated high expression level and the phosphorylation activation of RET, and experiment in vitro is found, many target drugs all can suppress the propagation of this cell as Xarelto, Sutent and ZD6474 (vandetanib).
CCDC6-RET is newfound fusion gene in non-smoking patients with lung adenocarcinoma, and information biology and experiment results show, exist 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 kinase activity district of extracellular domain and the RET of CCDC6 has been merged in this transgenation, in lung cancer sample, often expression is higher, and this may be the key point that causes lung cancer morbidity.
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 RET of detection 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 that realizes above-mentioned purpose is as follows:
Detect a PCR primer for RET fusion gene, described PCR primer is: for K15; The SEQ ID NO.1 of R12 and SEQ ID NO.4, for K16; The SEQ ID NO.1 of R12 and SEQ ID NO.5, for K22; R12 and K23; The SEQID NO.1 of R12 and SEQ ID NO.6, for K24; The SEQ ID NO.2 of R8 and SEQ ID NO.7, for K24; The SEQ IDNO.3 of R11 and SEQ ID NO.7 and/or for C1; The SEQ ID NO.1 of R12 and SEQ ID NO.8.
Another object of the present invention is to provide a kind of PCR test kit of the RET of detection fusion gene.
The technical scheme that realizes above-mentioned purpose is as follows:
Detect a PCR test kit for RET fusion gene, include above-mentioned described PCR primer.
In an embodiment, described test kit also includes positive reference substance therein, and described positive reference substance is: described positive reference substance is: for K15; R12 contain SEQ ID NO.1 and with the nucleic acid fragment of the sequence of SEQ ID NO.4 reverse complemental pairing, for K16; R12 contain SEQ ID NO.1 and with the nucleic acid fragment of the sequence of SEQ ID NO.5 reverse complemental pairing, for K22; R12 and K23; R12 contain SEQ ID NO.1 and with the nucleic acid fragment of the sequence of SEQ ID NO.6 reverse complemental pairing, for K24; R8 contain SEQ ID NO.2 and with the nucleic acid fragment of the sequence of SEQ ID NO.7 reverse complemental pairing, for K24; R11 contain SEQ ID NO.3 and with the nucleic acid fragment of the sequence of SEQ ID NO.7 reverse complemental pairing and/or for C1; R12's contains SEQ ID NO.1 and the nucleic acid fragment with the sequence of SEQ ID NO.8 reverse complemental pairing.
More preferably, described positive reference substance is: for K15; The SEQ ID NO.9 of R12, for K16; The SEQ IDNO.10 of R12, for K22; The SEQ ID NO.11 of R12, for K23; The SEQ ID NO.12 of R12, for K24; The SEQID NO.13 of R8, for K24; The SEQ ID NO.14 of R11 and/or for C1; The SEQ ID NO.15 of R12.
Another object of the present invention is to provide the liquid-phase chip that a kind of RET of detection fusion gene detects.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 that realizes above-mentioned purpose is as follows:
A liquid-phase chip that detects RET fusion gene, includes:
(A) above-mentioned PCR primer;
(B). the ASPE primer designing respectively for the different fused type of RET 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 K15; The SEQ ID NO.23 of R12, for K16; The SEQ ID NO.24 of R12, for K22; The SEQ ID NO.25 of R12, for K23; The SEQ ID NO.26 of R12, for K24; The SEQ ID NO.27 of R8, for K24; The SEQ ID NO.28 of R11 and/or for C1; The SEQ ID NO.29 of R12; Described tag sequence is selected from SEQ ID NO.16～SEQ ID NO.22;
(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.30～SEQ ID NO.36, and described anti-tag sequence can be correspondingly with (B) in selected tag sequence complementary pairing.
In an embodiment, described ASPE primer is: for K15 therein; The sequence being formed by SEQ ID NO.16 and SEQ IDNO.23 of R12, for K16; The sequence being formed by SEQ ID NO.17 and SEQ ID NO.24 of R12, for K22; The sequence being formed by SEQ ID NO.18 and SEQ ID NO.25 of R12, for K23; The sequence being formed by SEQ ID NO.19 and SEQ ID NO.26 of R12, for K24; The sequence being formed by SEQ ID NO.20 and SEQ ID NO.27 of R8, for K24; The sequence being formed by SEQ ID NO.21 and SEQ ID NO.28 of R11 and/or for C1; The sequence being formed by SEQID NO.22 and SEQ ID NO.29 of R12.
Another object of the present invention is to provide a kind of Auele Specific Primer for detection of RET fusion gene.
Concrete technical scheme is as follows:
For detection of an Auele Specific Primer for RET fusion gene, described Auele Specific Primer is for K15; The SEQ IDNO.23 of R12, for K16; The SEQ ID NO.24 of R12, for K22; The SEQ ID NO.25 of R12, for K23; The SEQ ID NO.26 of R12, for K24; The SEQ ID NO.27 of R8, for K24; The SEQ ID NO.28 of R11 and/or for C1; The SEQ ID NO.29 of R12.
Major advantage of the present invention is:
1, the identical rate of detected result that RET fusion gene provided by the present invention detects liquid phase chip reagent box and sequencing is 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 strict screening, can complete the amplification of the target sequence that contains 7 kinds of 1RT fusion gene hypotypes by a step PCR, 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 method 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 RET 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.
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 the fusion gene of RET fusion gene detects
One, RT-PCR amplification
1, reverse transcription
The RET 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 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 design forward primer and reverse primer.Use the cDNA of step 1 gained as template, to the amplification that walks abreast of 7 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/LTris Buffer.
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 kinds of hypotypes of target detect RET 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.9 ~ SEQ ID NO.15 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, 7 kinds of RET fusion gene hypotype recombinant plasmid dnas that establishing target detects are as positive reference substance.7 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 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, 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 7 kinds of fusion gene hypotypes, one step amplifies 7 target sequences that contain fusion gene hypotype, product size is as shown in table 1, and amplimer sequence (SEQ ID NO.1 ~ SEQ ID NO.8) 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.8 in 1.5ml Eppendorf tube, mix and be multiple PCR primer working fluid.Multi-PRC reaction system is 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, Fig. 1 is the electrophoresis picture that polyacrylamide gel detects 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 7 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 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 the specific primer sequence of totally 7 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 two kinds of microballoons numberings of selection and microballoon, corresponding anti-tag sequence is as shown in table 2:
Corresponding anti-tag sequence on table 4 microballoon numbering and microballoon
7 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/LTris(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 detection method of RT-PCR amplified production in a kind of embodiment of detection 1, use the detection method in embodiment 2 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 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 2, 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 7 kinds of fusion gene hypotypes, one step amplifies 7 target sequences that contain fusion gene hypotype, and product size is as shown in table 1.Primer sequence (SEQ ID NO.1 ~ SEQ IDNO.8) 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.8 in 1.5ml Eppendorf tube, mix and be multiple PCR primer working fluid.Multi-PRC reaction system is 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.
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 37 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 RET 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 RET fusion gene hypotype detected result and the sequencing result rate of coincideing originally and reaches 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) that prepared by 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) of these 3 kinds of fusion gene hypotype design ASPE primer 3 ' ends, the Tag sequence of ASPE primer 5 ' end is selected from SEQ ID NO.16-SEQ ID NO.22, accordingly, the anti-tag sequence with corresponding tag sequence complementary pairing being coated on microballoon is selected from SEQ ID NO.30-SEQ ID NO.36.Specific design is as shown in following table (table 7).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 7 liquid-phase chip
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 61-80 by testing process described in embodiment 2 and method, and 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- |
| 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 K15; R12, K16; R12, K22; R12, K23; R12, K24; R8, K24; R11, C1; R12 is the cross reacting rate of totally 7 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 106 copies/uL, loading 10uL repeats 3 times.
2, the mixed solution of positive control plasmid is carried out to pcr amplification, a step amplifies 7 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.8 in 1.5ml Eppendorf tube, mix and be multiple PCR primer working fluid.Multi-PRC reaction system is 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.
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)
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.
For 7 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 7 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:
PCR program is: 96 ℃ of 2min; 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ save backup.
Five, hybridization (as described in Example 4)
Six, result detects and data analysis
Reaction after product detects by Luminex serial analysis instrument.
Table 10 sample plasmid detected 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) that prepared by liquid-phase chip
With RET fusion gene K16; R12; K24; R8; C1; The detection liquid-phase chip of R12 hypotype is example, take the complementary sequence forward or backwards of this place, mutational site target sequence as template, respectively for K16; R12; K24; R8; C1; The specific primer sequence of R12 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 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 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 12).Synthetic, the anti-tag sequence of ASPE primer are coated with microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Design prepared by table 12 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 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- |
| 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 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, identical with the detected result of embodiment 4 and the present embodiment, 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 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 (8)
1. a PCR primer that detects RET fusion gene, is characterized in that, described PCR primer is: for K15; The SEQID NO.1 of R12 and SEQ ID NO.4, for K16; The SEQ ID NO.1 of R12 and SEQ ID NO.5, for K22; R12 and K23; The SEQ ID NO.1 of R12 and SEQ ID NO.6, for K24; The SEQ ID NO.2 of R8 and SEQ ID NO.7, for K24; The SEQ ID NO.3 of R11 and SEQ ID NO.7 and/or for C1; The SEQ ID NO.1 of R12 and SEQ ID NO.8.
2. a PCR test kit that detects RET 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, described test kit also includes positive reference substance, and described positive reference substance is: for K15; R12 contain SEQ ID NO.1 and with the nucleic acid fragment of the sequence of SEQ ID NO.4 reverse complemental pairing, for K16; R12 contain SEQ ID NO.1 and with the nucleic acid fragment of the sequence of SEQ ID NO.5 reverse complemental pairing, for K22; R12 and K23; R12 contain SEQ ID NO.1 and with the nucleic acid fragment of the sequence of SEQ ID NO.6 reverse complemental pairing, for K24; R8 contain SEQ ID NO.2 and with the nucleotide sequence of the sequence of SEQ ID NO.7 reverse complemental pairing, for K24; R11 contain SEQ ID NO.3 and with the nucleic acid fragment of the sequence of SEQ ID NO.7 reverse complemental pairing and/or for C1; R12's contains SEQ ID NO.1 and the nucleic acid fragment with the sequence of SEQ ID NO.8 reverse complemental pairing.
4. PCR test kit according to claim 3, is characterized in that, described positive reference substance is: for K15; The SEQ ID NO.9 of R12, for K16; The SEQ ID NO.10 of R12, for K22; The SEQ ID NO.11 of R12, for K23; The SEQ ID NO.12 of R12, for K24; The SEQ ID NO.13 of R8, for K24; The SEQ ID NO.14 of R11 and/or for C1; The SEQ ID NO.15 of R12.
5. the liquid-phase chip that RET fusion gene detects, is characterized in that, includes:
(A) PCR primer claimed in claim 1;
(B) the ASPE primer designing respectively for the different fused type of RET 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 K15; The SEQ ID NO.23 of R12, for K16; The SEQ ID NO.24 of R12, for K22; The SEQ ID NO.25 of R12, for K23; The SEQ ID NO.26 of R12, for K24; The SEQ ID NO.27 of R8, for K24; The SEQ ID NO.28 of R11 and/or for C1; The SEQ ID NO.29 of R12; Described tag sequence is selected from SEQ ID NO.16～SEQ ID NO.22;
(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.30～SEQ ID NO.36, and described anti-tag sequence can be correspondingly with (B) in selected tag sequence complementary pairing.
6. described liquid-phase chip according to claim 5, is characterized in that, described ASPE primer is: for K15; The sequence being formed by SEQ ID NO.16 and SEQ ID NO.23 of R12, for K16; The sequence being formed by SEQ ID NO.17 and SEQ IDNO.24 of R12, for K22; The sequence being formed by SEQ ID NO.18 and SEQ ID NO.25 of R12, for K23; The sequence being formed by SEQ ID NO.19 and SEQ ID NO.26 of R12, for K24; The sequence being formed by SEQ ID NO.20 and SEQ ID NO.27 of R8, for K24; The sequence being formed by SEQ ID NO.21 and SEQ ID NO.28 of R11 and/or for C1; The sequence being formed by SEQ ID NO.22 and SEQ ID NO.29 of R12.
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 RET fusion gene, it is characterized in that, described Auele Specific Primer is: for K15; The SEQ ID NO.23 of R12, for K16; The SEQ ID NO.24 of R12, for K22; The SEQ ID NO.25 of R12, for K23; The SEQ ID NO.26 of R12, for K24; The SEQ ID NO.27 of R8, for K24; The SEQ ID NO.28 of R11 and/or for C1; The SEQ ID NO.29 of R12.
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| CN107022610A (en) * | 2017-04-01 | 2017-08-08 | 常州桐树生物科技有限公司 | The detection chip of tumour driving gene and its application |
| 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 |
| CN110205364A (en) * | 2019-06-28 | 2019-09-06 | 北京和合医学诊断技术股份有限公司 | RET detection in Gene Mutation primer sets, kit, system and method |
| 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 |
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