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Primer and probe combination for detecting methylation of genes related to high-grade cervical lesions and cervical cancer

A high-level, methylation technology, applied in the field of biomedicine, can solve the problems of insufficient correlation of cervical cancer and achieve the effect of improving prediction accuracy, detection sensitivity and specificity

Active Publication Date: 2021-08-17
苏州凯爱健康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to solve the problem of insufficient correlation between existing methylation detection and cervical high-grade lesions and cervical cancer, to achieve more accurate and effective cervical high-grade lesions and cervical cancer auxiliary diagnosis, shunt management and prognosis judgment technology, to improve cervical cancer The efficiency of cancer screening, the invention provides a primer, probe, PCR amplification reagent for detecting human genome DNA methylation and HPV virus DNA methylation, and a method for analyzing the risk of cervical high-grade lesions and cervical cancer ;

Method used

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  • Primer and probe combination for detecting methylation of genes related to high-grade cervical lesions and cervical cancer
  • Primer and probe combination for detecting methylation of genes related to high-grade cervical lesions and cervical cancer
  • Primer and probe combination for detecting methylation of genes related to high-grade cervical lesions and cervical cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 Reaction Steps and Reaction System for Detection of High-grade Cervical Lesions and Cervical Cancer According to the Present Invention

[0093] a) Collection of biological samples:

[0094] The cervical cells brushed off by the cervical cell brush are stored in the cell preservation solution.

[0095] Wherein said cell preservation solution can be sample transfer medium (Specimen Transport Medium, STM), careHPV collection medium (careHPV Collection Medium), SurePath cell preservation solution and PreservCyt cell preservation solution.

[0096] 2) Extraction of cellular DNA

[0097] Take 200 μL of the cell preservation solution containing cervical exfoliated cells as a sample, and use the Magnetic Bead Method Animal Tissue Genomic DNA Extraction Kit and Nucleic Acid Extraction Kit provided by Tiangen Biochemical Technology Co., Ltd. for DNA extraction and purification.

[0098] 3) DNA transformation

[0099] DNA extracted from cells was used as a sample, and...

Embodiment 2

[0117] Example 2 Screening and verification of primer probes for cervical high-grade lesions and cervical cancer detection according to the present invention

[0118] 1) Design of primers and probes

[0119] For human EPB41L3 gene 425, 427, 438 sites and internal reference ACTB gene (see NCBI database number NC_00018.10 and NC_000007.14 for sequence), and virus HPV16L1 gene 6367, 6389 site (sequence see NCBI database number is AF125673.1 ) and HPV18L2 gene 4256, 4261, 4265, 4269, 4275 and 4282 sites (see NCBI database number AY262282.1 for sequence), use Primer Premier 5.0 and Methy Primer Express v1.0 software to design specific primers and probes respectively .

[0120] Primers and probes are shown in SEQ ID No: 1-53; specific primers and probe names, nucleotide sequences and combination names are shown in Table 4 below:

[0121] The name, nucleotide sequence and combination of table 4 primers and probes

[0122]

[0123]

[0124] 2) Selection of positive control su...

Embodiment 3

[0167] Example 3 Performance Verification of Cervical High-grade Lesions and Cervical Cancer Detection According to the Present Invention

[0168] 1) Design of primers and probes

[0169] The optimized specific primers and probes were screened according to the above method, the internal reference ACTB primer probe combination 1, the combination of EPB41L3 primer probe, HPV16L1 primer probe, HPV16 internal reference primer probe, HPV18L2 primer probe and HPV18 internal reference primer probe 1 and combination 3 are divided into two groups for combination:

[0170] Combination 1: internal reference ACTB primer probe combination 1, combination 1 of EPB41L3 primer probe, HPV16L1 primer probe, HPV16 internal reference primer probe, HPV18L2 primer probe and HPV18 internal reference primer probe;

[0171] Combination 3: Internal reference ACTB primer probe combination 1, combination 3 of EPB41L3 primer probe, HPV16L1 primer probe, HPV16 internal reference primer probe, HPV18L2 prime...

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Abstract

The invention discloses a primer and a probe for detecting methylation degrees of genes related to high-grade cervical lesions and cervical cancer. The primer and the probe are used for detecting methylation of sites 425, 427 and 438 of the human EPB41L3 gene, methylation of sites 6367 and 6389 of the viral HPV16L1 gene and methylation of sites 4256, 4261, 4265, 4269, 4275 and 4282 of the HPV18L2 gene. Through a large amount of screening and optimization in the invention, the detection limits of the finally obtained methylation-specific primer and probe on the EPB41L3 gene, the HPV16L1 gene and the HPV18L2 gene are all within 1%. According to the invention, the problem that an existing methylation detection result is insufficient in correlation with high-level cervical lesions and cervical cancer is solved, a more accurate and effective technology for auxiliary diagnosis, scattered management and prognosis judgment of the high-level cervical lesions and cervical cancer is achieved, and cervical cancer screening efficiency is improved.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a combination of primers and probes for detecting high-grade cervical lesions and methylation of cervical cancer-related genes, specifically, specific primers and probes for methylation of EPB41L3, HPV16L1, and HPV18L2 and their respective internal reference gene primers and probes. Background technique [0002] Cervical cancer is one of the most common female malignant tumors. Human papillomavirus (HPV) is detected in cervical exfoliated cells of 99% of cervical cancer patients. Therefore, high-grade cervical lesions and cervical cancer are diseases with a clear etiology. Early prevention and early intervention can be achieved; in HPV infection, 91% of HPV virus is a transient infection and can be cleared by the autoimmune system, normal cervical cells develop from precancerous lesions to invasive cancer with a latent period of 5 to 10 years Therefore, early dete...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6886C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6883C12Q1/6886C12Q1/708C12Q1/6851C12Q2600/154C12Q2600/166C12Q2531/113C12Q2545/101C12Q2523/125C12Q2563/107
Inventor 石立立范贝贝韩亮陈华邢星伟柴银玲
Owner 苏州凯爱健康科技有限公司
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