Primer and kit for detecting septin9 gene methylation

A methylation and kit technology, applied in the field of gene septin9 methylation detection, can solve the problems of low detection sensitivity and specificity, and achieve the effects of increased sensitivity and specificity, high sensitivity and easy interpretation

Inactive Publication Date: 2016-12-21
北京鑫诺美迪基因检测技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problem of low sensitivity and specificity in the detection of the methylation of the colorectal cancer-related gene septin9 gene in the prior art,

Method used

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  • Primer and kit for detecting septin9 gene methylation
  • Primer and kit for detecting septin9 gene methylation
  • Primer and kit for detecting septin9 gene methylation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 kit preparation

[0036] Primer and probe sequences contained in the kit:

[0037] Septin9 forward primer F: 5'-CCGAAATAATCCCATCCAAC-3',

[0038] Septin9 reverse primer R: 5'-GCGATTCGTTGTTTATTAGTTATTAT-3';

[0039] Internal reference gene forward primer F: 5'-GTGATGGAGGAGGTTTAG-3',

[0040] Internal reference gene reverse primer R: 5'-AAATTACAAAAACCACAA-3';

[0041] septin9 gene probe: FAM-TTATGTCGGATTTCGCGGTTAA-MGB-BHQ1;

[0042] Internal reference gene probe: VIC-CACCACCCAAACACACAATAACAAAC-BHQ1;

[0043] Blocker1 primer: 5'-ACCCGCTGCCCACCAGCCATCATG-CY3-3';

[0044] Blocker2 primer: 5'-TATGTTGGATTTTGTGGTTAATGTGTA-CY3-3'.

[0045] (1) Premixed primer-probe mixture for detecting septin9 gene methylation:

[0046]

[0047] (2) DNA PCR amplification MIX:

[0048]

Embodiment 2

[0049] Embodiment 2 kit detects septin9 gene methylation

[0050] 1) 10 cases of plasma samples were taken for free DNA extraction, and the free DNA extraction kit was from Tiangen Biochemical Technology Co., Ltd.;

[0051] 2) The extraction of free DNA is completed, and the extracted DNA is converted to bisulfite to obtain purified DNA. The conversion kit is from Tiangen Biochemical Technology Co., Ltd.;

[0052] 3) Prepare PCR amplification reaction system:

[0053]

[0054]

[0055] 4) The PCR amplification procedure is as follows:

[0056] The first amplification stage: UDG enzyme reaction at 37°C for 2 minutes;

[0057] The second amplification stage: pre-denaturation at 95°C for 3 minutes;

[0058] The third amplification stage: denaturation at 94°C for 15s, annealing and extension at 60°C for 35s, 45 cycles;

[0059] Signal collection, in the third stage, FAM and VIC signals were collected at 60°C.

[0060] 5) Analysis of test results

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Abstract

The invention discloses a primer and a kit for detecting septin9 gene methylation. The primer comprises a target gene primer pair Septin9 forward primer F and an Septin9 reverse primer R. The kit comprises the primer and optimally further comprises a reference gene primer, a TaqMan-MGB probe and two Blocker primers. The kit is simple and convenient to operate, is easy to judge and is low in requirement for instruments; the whole PCR process is fully closed; the cross contamination possibility is avoided; the result is more accurate. Compared with the method of detecting by adopting PCR specific primer, the method of identifying the methylated site by utilizing the probe has higher flexibility and accuracy. Two Blocker primers are utilized to strictly control the amplification of the non-specific stripe, so that the kit has higher detection sensitivity and specificity and is especially fit for early screening of free DNA of colorectal cancer plasma.

Description

technical field [0001] The invention relates to the technical field of gene diagnosis of diseases, in particular to the detection of methylation of a gene septin9 related to colorectal cancer. Background technique [0002] Colorectal cancer is the third most common cancer in the world, with 600,000 deaths per year due to colorectal cancer. The 5-year survival rate of patients with early colorectal cancer is about 80%. Since colorectal cancer often has no special symptoms in the early stage, it is easy to delay the timing of treatment, and the survival rate in the advanced stage is only about 10%. Before 2000, some countries in Europe and the United States had promoted the screening of high-risk groups for colorectal cancer throughout the country. In 2006, 60% of people aged 50 to 75 in the United States participated in at least one colorectal cancer screening. In recent years, the incidence of colorectal cancer among malignant tumors in my country ranks third, as high as 3...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2531/113C12Q2545/101C12Q2561/101
Inventor 刘沛王林海
Owner 北京鑫诺美迪基因检测技术有限公司
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