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

A fusion gene and liquid phase chip technology, which is applied in the field of molecular biology, can solve the problems of inaccurate identification of fusion types, various types of reagents, and cumbersome test procedures, so as to improve detection accuracy, increase fluorescence signal value, and overcome sensitivity. less sexual effect

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 A kind of PCR primer and positive control substance for detection of ROS1 fusion gene detection

[0041] 1. RT-PCR amplification

[0042] 1. Reverse transcription

[0043] For the ROS1 fusion gene subtypes detected by various targets, the present invention first uses random primers to perform reverse transcription on the target detection samples, 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.

[0044] 2. PCR amplification

[0045] The fusion types in Table 1 are the 11 ROS1 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, 11 target detection fusion gene subtypes were a...

Embodiment 2

[0055] Example 2 uses the PCR primers in Example 1 to detect the ROS1 fusion gene

[0056] 1. PCR amplification

[0057] Using the PCR amplification primers described in Table 1 in Example 1, PCR amplification was carried out to the mRNA reverse transcription product of the target detection sample and the positive control plasmid mixture, and at the same time, in order to facilitate the use of conventional electrophoresis techniques to distinguish PCR amplification bands , according to the size of the PCR product (as shown in Table 1), the 11 fusion types detected by the target and their corresponding positive control plasmids were divided into 2 groups, and 2 independent PCR amplifications were carried out at the same time, and agarose gel was used to Gel and polyacrylamide gel for detection, according to the size of the amplified product, PCR primers are divided into required categories, and detected by electrophoresis technology, which is a conventional method in the art. ...

Embodiment 3

[0068] Example 3 A liquid chip for detecting ROS1 fusion gene

[0069] 1. ASPE Primers

[0070] R32, C6; R34, C5; R34; S2; R32, S4; R32, S4; R34 of SDC4-ROS1; SL13; R32, SL4; R34 of SLC34A2-ROS1 ; E10 of EZR-ROS1; R34; L16 of LRIG3-ROS1; R35 and T5 of TPM3-ROS1; R35, a total of 11 specific primer sequences designed for fusion subtypes. ASPE primers consist of "Tag + specific primer sequence". ASPE primer sequences are shown in the table below:

[0071] Table 3 ASPE primer sequence ((Tag + specific primer sequence))

[0072]

[0073] 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.

[0074] 2. Microspheres coated with anti-tag sequences

[...

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Abstract

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

Description

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 detecting ROS1 fusion gene. technical background [0002] v-ros UR2 sarcoma viral oncogene homolog 1 (v-ros avian UR2 sarcoma viral oncogene homolog 1, ROS1), in 1987 Rabin M. cloned the gene related to the growth of v-ros sarcoma and named it ROS gene. There are breakpoints at the 32nd, 34th and 35th exons of the ROS1 gene, which can be fused with various genes. The ROS1 fusion gene was confirmed as a new lung cancer driver gene, which plays an important role in the formation of lung adenocarcinoma. At the 48th annual meeting of the American Society of Clinical Oncology (ASCO) held in June 2012, Shaw of the Massachusetts General Hospital reported for the first time the phase I clinical trial of patients with ROS1 gene fusion, a new molecular target for lung cancer. The clinical trial proved t...

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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