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PCR (Polymerase Chain Reaction) primer, kit and liquid chip for detecting RET fusion gene

Active Publication Date: 2014-05-21
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The RT-PCR method is to design primers for the known RET fusion gene types and fusion methods. After the RNA is reverse-transcribed to obtain cDNA, the target fragment is amplified by PCR for detection, which has low sensitivity and easy contamination of the sample. , the disadvantage of high false positive rate
Although the non-radioactive in situ hybridization technique FISH method is relatively intuitive, the test process is too cumbersome, requires a wide variety of reagents, is time-consuming and laborious, and it can only detect whether the fusion gene exists, and cannot accurately determine the specific fusion type. Sensitivity low, thus limiting the application of the law to a certain extent

Method used

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  • PCR (Polymerase Chain Reaction) primer, kit and liquid chip for detecting RET fusion gene
  • PCR (Polymerase Chain Reaction) primer, kit and liquid chip for detecting RET fusion gene
  • PCR (Polymerase Chain Reaction) primer, kit and liquid chip for detecting RET fusion gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 A kind of PCR primer and positive control substance detected by the fusion gene detection of RET fusion gene

[0034] 1. RT-PCR amplification

[0035] 1. Reverse transcription

[0036] For the subtypes of RET fusion genes detected by various targets, the present invention uses random primers to reverse-transcribe the target detection samples first, and reverse-transcribes the mRNA in the samples into cDNA. The specific experimental steps refer to the "Molecular Cloning Experiment Guide", using random primers Reverse transcription of mRNA is a routine procedure.

[0037] 2. PCR amplification

[0038] The fusion types in Table 1 are the seven RET fusion gene subtypes detected by the target of the present invention. According to the nucleic acid composition characteristics of the fusion gene sequence, the forward primer and reverse primer were designed using Primer5.0. Using the cDNA obtained in step 1 as a template, the seven target detection fusion gene s...

Embodiment 2

[0050] Example 2 uses PCR primers in Example 1 to detect the RET fusion gene

[0051] 1. PCR amplification

[0052] Using the PCR amplification primers described in Table 1, the mRNA reverse transcription product of the target detection sample and the positive control substance were amplified by PCR to realize the parallel amplification of the target sequences of the 7 fusion gene subtypes, and 7 fusion gene subtypes were amplified in one step. The strip contains the target sequence of the fusion gene subtype, the product size is shown in Table 1, and the amplification primer sequences (SEQ ID NO.1~SEQ ID NO.8) are shown in Table 1 above.

[0053] First, prepare the PCR primer working solution: take 100ul of the primer stock solutions of SEQ ID NO.1~SEQ ID NO.8 respectively in 1.5ml microcentrifuge tubes, and mix well to obtain the multiplex PCR primer working solution. The multiplex PCR reaction system is as follows:

[0054]

[0055] The PCR amplification program is: 95...

Embodiment 3

[0059] Example 3 A liquid-phase chip kit for detecting RET fusion gene

[0060] 1. ASPE Primers

[0061] Specific primer sequences designed for seven fusion subtypes of RET fusion genes: K15; R12, K16; R12, K22; R12, K23; R12, K24; R8, K24; R11, C1; R12. ASPE primers consist of "Tag + specific primer sequence". ASPE primer sequences are shown in the table below:

[0062] Table 3 ASPE primer sequence (Tag+ specific primer sequence)

[0063]

[0064] Each ASPE primer consists of two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer sequence (as shown in Table 3 above). All ASPE primers were synthesized by Invitrogen. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.

[0065] 2. Microspheres coated with anti-tag sequences

[0066] According to the designed ASPE-specific primer sequence, select the tag sequence ...

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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 [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a PCR primer, a kit and a liquid phase chip for RET fusion gene detection. technical background [0002] Chromosomal rearrangements in cancer often result in the fusion of two separate genes. In some cases, the coding regions of two genes are linked together, resulting in the expression of fusion proteins, known as gene fusions. In mammals, gene fusion is one of the causes of certain diseases such as cancer. Gene fusion was first discovered in various blood and soft tissue cancers, such as leukemia and lymphoma. RET proto-oncogene is located on autosome 11q11.2, about 60000bp in length, contains at least 20 exons, and encodes a tyrosine kinase receptor. Current studies have shown that RET gene mutations are closely related to the occurrence of diseases such as fMT C, sMT C, multiple endocrine tumors 2A and 2B, and Hirschsprung's disease. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C40B40/06
CPCC12Q1/6827C12Q1/686C12Q2563/149C12Q2531/113
Inventor 陈昌华陈菲许昌有
Owner SUREXAM BIO TECH
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