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Primer pair, kit and method based on cervical cancer specificity methylation detection

A specificity and methylation technology, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome operation, low accuracy and low sensitivity, and save society resource, fast and accurate measurement, good effect of sensitivity and specificity

Inactive Publication Date: 2017-05-31
韩林志
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] In order to solve the technical problems of cumbersome operation, low accuracy and low sensitivity in the above-mentioned traditional methylation detection method, the present invention provides a cervical cancer-specific detection method based on fluorescent PCR technology that is simple to operate, high in accuracy and high in sensitivity. Primer pair, kit and method for sex methylation detection

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  • Primer pair, kit and method based on cervical cancer specificity methylation detection
  • Primer pair, kit and method based on cervical cancer specificity methylation detection
  • Primer pair, kit and method based on cervical cancer specificity methylation detection

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1: the preparation of kit

[0040] 1. Design and synthesis of primers and probes

[0041] For the promoter regions of the PAX1 gene and the internal standard gene ACTB (β-actin) in the human genome (for the sequence, refer to the human whole genome sequence published in the NCBI database), use Primer Premier 3.0 and Methyl Primer Expressv1.0 software to design a pair of specific Primers and probes.

[0042] Specific primers and probe sequences are shown in the table below:

[0043]

[0044] 2. Selection of reference substance

[0045] Use methylated standard DNA as a positive control, its sequence is:

[0046]AAATTGATTttCGtACGtTGtAGttTttCGGTtAGACGAATTTtTtttAATCGGATGAAGTTtAtttTGGGttTGGGGTCGCGGGCGTGGAGAGTGTttTGGGAGGGGGtAGtAGCGGCGGCGGtAGGtttTGGAGCGGGCGGtAGCGCGtTtCGtTGtCGCGtAtAGCGCGTtTttAGttCGCGGtTGGGtCGtCGCGGtTtTCG;

[0047] Non-methylated standard DNA is a negative control, and its sequence is:

[0048] AAATTGATTtttGtAtGtTGtAGttTtttGGTtAGAtGAATTTtTtttAA...

Embodiment 2

[0070] Example 2: Method for detecting cervical cancer methylation using the above kit

[0071] 1. Technical principle

[0072] A pair of specific primers and probes for methylation detection were designed respectively in the promoter regions of the PAX1 gene and the internal standard gene ACTB (b-actin) in the human genome. Then use the primers and probes to amplify the sample DNA converted by sulfite, and determine the methylation rate of the sample to be tested according to the relative fluorescence CT value of the PCR amplification results of PAX1 and ACTB genes. Assess the risk of cervical cancer.

[0073] 2. Detection method

[0074] Step 1: Take the DNA extracted from the sample to be tested, carry out transformation treatment on it, and use the transformed DNA as a template for PCR;

[0075] Wherein, the reagents used in the conversion treatment are bisulfite or bisulfite, and other auxiliary materials (the corresponding kit is EpiTect Fast DNA BisuLfite Kit purchas...

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Abstract

The invention relates to a primer pair based on cervical cancer specificity methylation detection. The primer pair comprises a PAX1 forward primer, a reverse primer and a detection probe as well as an ACTB forward primer, a reverse primer and a detection probe. The invention also relates to a kit based on cervical cancer specificity methylation detection. The kit comprises PCR reaction liquid containing the primers and the probes, including the PAX1 forward primer, the PAX1 reverse primer, the PAX1 detection probe, the ACTB forward primer, the ACTB reverse primer, the ACTB detection probe, 10*PCR buffer, dNTP, nuclease-free water and Ex Taq enzyme. The invention also relates to a method based on cervical cancer specificity methylation detection. The kit and the detection method thereof provided by the invention have the advantages of accurate detection result, high detection throughput, good specificity and sensitivity, fast detection and convenience in use and can effectively meet clinical requirements.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnosis, in particular, to a primer pair, a kit and a method based on cervical cancer-specific methylation detection. Background technique [0002] Cervical cancer is one of the most common gynecological malignancies and a major disease that endangers women's health and life. Epidemiological surveys show that there are 465,000 new cases of cervical cancer in the world every year, of which about 130,000 are new cases in China every year. At present, the age of onset of cervical cancer tends to be younger, and women affected by cervical cancer are mostly in the golden age of childbearing and family management. [0003] Cervical cancer is different from other tumors. The natural history of the disease is clear, and there are a series of precancerous lesions. Its occurrence and development are from quantitative change to qualitative change, gradual change to mutation, and it takes several years to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/154C12Q2600/166
Inventor 韩林志肖芳李书
Owner 韩林志
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