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Primers, kit and method for detecting SYT-SSX fusion genes

A technology of SYT-SSX1 and SYT-SSX-F, applied in the field of molecular biology, can solve the problems of poor specificity and high cost, and achieve the effects of high sensitivity, increased specificity, and strong specificity

Pending Publication Date: 2019-09-10
杭州艾迪康医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, because SYBR GreenI is an unsaturated dye, its specificity is not as good as that of the double-probe hybridization method and the Taqman method, and its specificity must be judged by observing the melting curve; and the cost of the double-probe hybridization method is relatively expensive.

Method used

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  • Primers, kit and method for detecting SYT-SSX fusion genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] ARMS-QPCR kit for detecting SYT-SSX fusion gene, including qPCR reaction system, qPCR reaction system includes qPCR mixed reaction solution, reaction primers and positive control samples;

[0065] qPCR mixed reaction solution includes PCR buffer, dNTP, MgCl 2 , KOD enzyme, PCR upstream and downstream primers and TaqMan probes, primers and probes for detecting internal reference genes, and positive control samples; in the qPCR reaction system, the final concentrations of each component are: PCR buffer is 1X; dNTP is 1.5mM ; MgCl 2 2mM; KOD enzyme 0.6U / μl; PCR upstream and downstream primers 0.1μM; TaqMan probe 0.1μM; positive control sample 1ng / μl;

[0066] The sequences of PCR upstream and downstream primers and TaqMan probes are:

[0067] SYT-SSX-F: 5'-CACCACAGCCACCCCAGC-3'

[0068] SYT-SSX-TaqMan: 5'-FAM ACCAGATCATGCCCAAAGCAGCCAGC-TAMRA-3';

[0069] SSX1-R: 5'-CACTCCCTTCGAATCATTTTCG-3';

[0070] SSX2-R: 5'-CACTTCCTCCGAATCATTTCCT-3';

[0071] Primers and probes f...

Embodiment 2

[0077] Detection process:

[0078] (1) Extract tissue RNA from blood.

[0079] (2) Reverse transcription: Refer to the instructions of the Rever Tra Ace qPCR RT Kit kit from TOYOBO, and reverse-transcribe the tissue RNA in (1) into cDNA.

[0080] (3) Reagent configuration: configure fusion gene detection qPCR reaction solution and internal reference gene detection qPCR reaction solution according to the number of people to be tested;

[0081] (4) Adding samples: Take 2ul of the cDNA obtained in step (2) and add them to the fusion gene detection qPCR reaction solution and the internal reference gene detection qPCR reaction solution configured in (3); for the positive control experiment, directly add 2ul positive control substance; for negative control substance experiment, directly add 2ul negative control substance; for blank control substance experiment, add 2ul normal saline or no substance.

[0082] (5) Detection: detection is carried out on a real-time fluorescent PCR in...

Embodiment 3

[0089] The nucleic acid detection kit of the present invention is used to detect clinical blood samples.

[0090] Take 20 cases of clinical blood samples to be tested, extract tissue RNA in the blood samples according to the detection process described in Example 2, reverse transcribe the tissue RNA into cDNA, prepare reagents and detect.

[0091] For each sample, 2ul of the obtained cDNA was added to the SYT-SSX fusion gene detection qPCR reaction solution and the internal reference gene detection qPCR reaction solution. Carry out positive, negative and blank control experiments at the same time.

[0092] Optimization of the reaction system:

[0093] (1) Optimization of the primer concentration. Under the same conditions in the reaction system, the primer concentration was serially diluted from 0.05 μM / L to 1.5 μM / L, and the optimal concentration was determined to be 0.1 μM through the analysis of the experimental results. / L.

[0094] (2) Optimization of the probe concentra...

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Abstract

The invention discloses primers, kit and method for detecting SYT-SSX fusion genes. The primers comprise the primer and a probe which are used for detecting an SYT-SSX1 or SYT-SSX2 fusion gene and theprimer and a probe which are used for detecting reference genes. Compared with direct sequencing and other technologies, the primers, kit and method for detecting the SYT-SSX fusion genes have the advantages that the specificity is strong, the sensitivity is high, the operation is simple and rapid, the throughput is high, interpretation results are reliable, and simpleness and convenience are achieved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an ARMS-qPCR primer, a kit and a method for detecting a SYT-SSX fusion gene, which can quickly, sensitively and accurately detect the SYT-SSX fusion gene. Background technique [0002] Synovial sarcoma is a relatively common soft tissue malignancy, accounting for 5%-10% of soft tissue sarcomas. There are four histological types: monophasic, biphasic, monophasic epithelial, and poorly differentiated [1]. The morphological changes of synovial sarcoma are complex, and it is necessary to rely on multiple samples and find the characteristics of biphasic differentiation of the tumor before the diagnosis can be made. Typical cases of synovial sarcoma, such as young and middle-aged patients, occur in tumors near the large joints of the limbs. X-ray examination shows different degrees of calcification. Combining with biopsy, it is found that the tumor has the characteristics of biphasic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2535/137C12Q2531/113C12Q2545/101C12Q2545/113C12Q2561/101C12Q2563/107
Inventor 王淑一吴鹏飞裘振亚
Owner 杭州艾迪康医学检验中心有限公司
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