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Kit for detecting gene methylation of cervical cells based on fluorescent quantitative PCR technology and application of kit

A methylation and fluorescent reporter group technology is applied in the field of nucleic acid combinations and kits for detecting cervical cell gene methylation, and achieves the effects of rapid and objective detection results, high sensitivity and cost savings

Active Publication Date: 2021-12-21
常州国药医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently no technical products on this technology platform that can quickly, easily and specifically detect these methylated genes. In order to further improve the screening efficiency of cervical cancer, explore and develop more specific, effective, more convenient and easier to apply in clinical practice. Molecular detection products are of great significance

Method used

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  • Kit for detecting gene methylation of cervical cells based on fluorescent quantitative PCR technology and application of kit
  • Kit for detecting gene methylation of cervical cells based on fluorescent quantitative PCR technology and application of kit
  • Kit for detecting gene methylation of cervical cells based on fluorescent quantitative PCR technology and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 detection kit and using method thereof

[0037] The kit includes the following components:

[0038] PCR reaction solution, enzyme mixture, detection reaction solution, positive control, negative control;

[0039]The PCR reaction solution includes 10× buffer, 25mM MgCl2, 10mM dUTP and 10mM dNTPs; the enzyme mixture includes Taq enzyme and UNG enzyme, and detects cervical cancer methylation genes SOX1, PAX1, MSC upstream and downstream primers, and probes in the reaction solution. The needle ratio is: 4:4:1; the upstream primer is 400nM, the downstream primer is 400nM, and the probe is 100nM.

[0040] See Table 1 for the nucleotide sequences of the primers and probes of the cervical cancer methylation gene SOX1 or PAX1 or MSC used in this example.

[0041] Table 1 Nucleotide sequence list of primers and probes of cervical cancer methylation gene SOX1 or PAX1 or MSC in the present invention

[0042] SOX1-F ATAAACTACCCGCTACCTACCCG SEQ ID No.1...

Embodiment 2

[0071] Embodiment 2: test kit detection accuracy

[0072] The 308 clinical samples that have been detected so far are compared with the gold standard method of cervical cancer-colposcopy combined with pathological detection results. There are 50 normal samples of pathological diagnosis, 47 cases of cervical invasive cancer samples, and 55 cases of carcinoma in situ CIS samples. , there are 115 cases of CIN3 stage samples, and 41 cases of CIN2 and below samples; the implementation method provided in Example 1 is used for detection. The comparison results of different stages can be seen in Table 6.

[0073] Table 6 Comparison of detection results of cervical cancer and precancerous lesions at different stages by multi-gene combined detection technology for cervical cancer

[0074]

[0075] Cervical exfoliated cell methylation gene (SOX1, PAX1, MSC) DNA detection result shows that the detection sensitivity of the test kit that embodiment 1 provides is respectively 97.39% and ...

Embodiment 3

[0076] Example 3: ROC Curve of Precancerous Lesions and Cervical Cancer

[0077] This experimental example provides the ROC curve for the detection of precancerous lesions and cervical cancer by the multi-target detection technology of exfoliated cell DNA.

[0078] In this experimental example, precancerous lesions and cervical cancer are detected experimentally by using the kit and experimental method provided in Example 1.

[0079] Tutu Institute figure 2 As shown, cervical exfoliated cell DNA multigene detection technology can clearly distinguish cervical precancerous lesions and cervical cancer from normal controls, and the area of ​​the ROC curve (AUC) for detecting CIN3 and CIS is 0.966 (0.95-0.98, 95%CI ; P<0.0001).

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Abstract

The invention relates to the technical field of biomedical examination, in particular to a kit for detecting gene methylation of cervical cells based on a fluorescent quantitative PCR technology and an application. According to the invention, whether a sample has canceration or not is judged by detecting the methylation level of promoter regions of an SOX1 gene, a PAX1 gene and an MSC gene in the sample. The kit is convenient and simple to operate, high in detection sensitivity and good in specificity, and has very positive significance in detection of cervical cancer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid combination, kit and application for detecting cervical cell gene methylation based on fluorescent quantitative PCR technology. Background technique [0002] Cervical cancer is the second most common cancer affecting women. Although HPV vaccination and early cervical cancer screening have become effective strategies to prevent the disease, high morbidity and mortality still exist. Persistent infection with high-risk human papillomavirus (HPV) is considered to be the main reason for the occurrence and development of cervical cancer. At present, the main method of clinical cervical cancer screening is cervical liquid-based cytology examination combined with HPV virus detection, but cytology detection is greatly influenced by human beings, and the probability of missed diagnosis is high. The limitations of HPV detection are as follows: (1 ) The lifetime risk of high-ris...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/154C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 李万帅孔祥宾汤丽丽
Owner 常州国药医学检验实验室有限公司
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