Kit for rapid detection of methylation of cervical exfoliated cells and detection method thereof

Abstract

The invention discloses a kit for rapid methylation detection of cervical exfoliated cells and a detection method thereof. The kit comprises a primer probe composition, an enzyme digestion amplification buffer solution, methylation sensitive restriction endonuclease and nuclease-free water, the primer probe composition comprises a ZNF671 primer and a probe, a PAX1 primer and a probe, an FAM19A4 primer and a probe, and an ACTIN primer and a probe, the kit is used for detecting the cervical exfoliated cells, the sensitivity and the specificity of a detection result are good, the detection process is simple, and consumed time is short.

Description

technical field [0001] The invention belongs to the technical field of methylation detection, in particular to a kit for rapid detection of methylation in cervical exfoliated cells and a detection method thereof. Background technique [0002] Cervical cancer is the most common gynecological malignancy. Persistent infection with high-risk human papillomavirus (HPV) is closely related to the occurrence of cervical cancer. After normal cervical cells are infected with HPV virus, most HPV viruses can be cleared by the human immune system , Only a small number of women will cause cervical precancerous lesions and develop cervical cancer due to persistent infection. [0003] At present, cytological detection methods and HPV-DNA detection are mainly used in the initial screening of cervical lesions; cytological detection methods have been used for more than half a century, including Pap smear method and liquid-based cytology developed in the past ten years (TCT), since the entire ...

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Problems solved by technology

[0003] At present, cytological detection methods and HPV-DNA detection are mainly used in the initial screening of cervical lesions; cytological detection methods have been used for more than half a century, including Pap smear method and liquid-based cytology developed in the past ten years (TCT), since the entire detection process is from sampling-smear preparation-observation-result interpretation, many links are affected by humans, so the probability of missed detection is high; HPV-DNA detection is only the presence or absence of HPV virus , and the presence of HPV virus is mostly transient, which means that these HPV infections that are cleared by autoimmunity will also be judged as positive, leading to overtreatment and colposcopy referral

[0004] In addition to the above two methods, methylation detection is also commonly used for the initial screening of cervical lesions, and the commonly used methods are methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), and methylation-sensitive PCR (BSP). Resolution melting curve analysis (MS-HRM), fluorescence quantitative method, and gene chip method are all methylation detection technologies based on sulfite modification pretreatment. The principle is that after DNA reacts with sodium bisulfite, no methylation Methylated cytosine (C) can be converted to uracil (U), while methylated cytosine shows no structural change, Methylated cytosine is determined by distinguishing the structural difference between uracil and methylated cytosine pyrimidines; although the BC strategy provides excellent performance in methylation detection, it still has disadvantages such as cumbersome pretreatment, time-consuming process, the need for multiple reagents, and extreme experimental conditions (such as strong acid, strong base, and high temperature), and more Unfortunately, the BC strategy suffers from target degradation, low DNA recovery, and incomplete conversion, leading to inaccurate measurements and poor reproducibility of methylation assays, especially in samples with low methylation abundance

[0005] Therefore, there is an urgent need to explore more sensitive and rapid methylation analysis methods. Methylation-sensitive restriction enzymes (MSREs) are a class of bacterial restriction enzymes that can specifically cut unmethylated CpG dinucleotides in the active site of the enzyme. , leaving only methylated DNA as an intact template for PCR or qPCR; this method can be used to determine the frequency of CpG methylation in any specific region of interest amplified by PCR, compared to the sulfite detection method, The detection operation has been simplified to a certain extent, but the detection time has not been significantly improved

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