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CN-121975937-A - Gene methylation primer probe composition, kit, application and diagnosis model for assisting in identifying cervical lesions

CN121975937ACN 121975937 ACN121975937 ACN 121975937ACN-121975937-A

Abstract

The invention discloses a group of gene methylation primer probe compositions, a kit, application and a diagnosis model for assisting in identifying cervical lesions, and relates to the technical field of biological detection, wherein the primer probe compositions are related to six genes of PAX1, SOX1, EPB41L3, ST6GALNAC5, SOX14 and ZNF 671. The kit comprises a nucleic acid extraction or purification reagent, a methylation detection sample pretreatment reagent, a PCR reaction mixed solution, a primer probe mixed solution 1, a primer probe mixed solution 2 and yin-yang quality control. The composition and the kit are used for detecting cervical high-level intraepithelial neoplasia, cervical squamous carcinoma and cervical adenocarcinoma which are HPV negative or HPV positive. The invention can improve the sensitivity and specificity of detection, reduce the input amount of samples, realize the detection of multiple types of cervical cancer in one-tube sample in cervical cancer screening, and can detect cervical cancer of different tissue pathological types.

Inventors

  • LIU YIDING
  • LI JIAHAO
  • HE JINJIA

Assignees

  • 成都诺森医学检验有限公司

Dates

Publication Date
20260505
Application Date
20260409

Claims (8)

  1. 1. A set of gene methylation primer probe compositions for aiding in the identification of cervical lesions, wherein the primer probe compositions are associated with six genes, PAX1, SOX1, EPB41L3, ST6GALNAC5, SOX14, ZNF671, the primer probe compositions comprising: PAX1-qF:TTTGGAGCGGGCG; PAX1-qR :GCCCGAAAACCGAA; PAX1-Probe:GACCCAACCGCGAACTAAAAACG; SOX1-qF:GAGCGGGTATTGGC; SOX1-qR:AAAAATCAAACGACTCA; SOX1-Probe:TTAGTGTACGTCGCGGTCGAGA; EPB41L3-qF: TTTTACGAGGTGCG; EPB41L3-qR: AACCGCGCGACGCCG; EPB41L3-Probe:CGAGGTTTGGGGCGAGGC; ST6GALNAC5-qF:CGTTGAGAGATTACGAGGGTTC; ST6GALNAC5-qR:CGACCGCGACAAATCG; ST6GALNAC5-Probe:GCGAAACCCGAAAAACGCG; SOX14-qF:CGTGGGGGTTTTCGAC; SOX14-qR:TAAACTACGCGAAATC; SOX14-Probe:CGCGTTCGAGAAAGTTCG; ZNF671-qF:CGGAAGTGTTTGCGTTTTC; ZNF671-qR:ACACCCACCCGCGCGA; ZNF671-Probe:CGTTTGTCGTTTTCGGTAGTTGTTC; reference gene-qF: GGAATTTTGTAGGTTTTATTTG; reference gene-qR CCTACACCCACAACACTATCT; Reference gene-qProbe: TAAACACCTAATCAAAAAAACAAACACCA.
  2. 2. The set of gene-methylated primer probe compositions for aiding in the identification of cervical lesions according to claim 1, wherein the probe sequence is labeled with a fluorescent group and a quencher group.
  3. 3. The set of gene-methylated primer probe compositions for aiding in the identification of cervical lesions according to claim 2, wherein the fluorophore is one of FAM, HEX, VIC, ROX, JOE, CY, the fluorophore is labeled at the 5 'end or 5' end of the probe sequence at 1bp-5 bp bases.
  4. 4. The set of gene-methylated primer probe compositions for aiding in the identification of cervical lesions according to claim 2, wherein the quenching group is one of BHQ1, BHQ2, BHQ3, the quenching group being labeled at the 3 'end, or 1bp-5 bp bases at the 3' end, of the probe sequence.
  5. 5. A kit for assisting in identifying cervical lesions, comprising a nucleic acid extraction or purification reagent, a methylation detection sample pretreatment reagent, a PCR reaction mixture, a primer probe mixture 1, a primer probe mixture 2, and a yin-yang quality control, wherein the primer probe mixture 1 and the primer probe mixture 2 each comprise the primer probe composition of any one of claims 1 to 4.
  6. 6. Use of a primer probe composition according to any one of claims 1-4 for the preparation of a kit for diagnosis of HPV negative or HPV positive cervical high grade intraepithelial neoplasia, cervical squamous carcinoma, cervical adenocarcinoma.
  7. 7. A diagnostic model, wherein the diagnostic model provides a result interpretation after gene methylation detection using the kit of claim 5, and wherein the diagnostic model has the following formula: P=(e^Z) / (1-e^Z); Z=12.130-0.040*△Ct(PAX1)-0.125*△Ct(SOX1)-0.220*△Ct(EPB41L3)-0.052*△Ct(ST6GALNAC5)-0.371*△Ct(SOX14)-0.336*△Ct(ZNF671); delta Ct (gene) =gene Ct value-reference gene Ct value.
  8. 8. The diagnostic model of claim 7, wherein a P value of 0.46 or greater indicates that there is a potential for high-grade intraepithelial neoplasia, squamous carcinoma of the cervix, adenocarcinoma of the cervix, and wherein a P value of <0.46 indicates that there is a lower risk of high-grade intraepithelial neoplasia, squamous carcinoma of the cervix, adenocarcinoma of the cervix.

Description

Gene methylation primer probe composition, kit, application and diagnosis model for assisting in identifying cervical lesions Technical Field The invention relates to the technical field of biological detection, in particular to a group of gene methylation primer probe compositions, a kit, application and a diagnosis model for assisting in identifying cervical lesions. Background More than about 95% of cervical cancers are caused by persistent infections with high risk human papillomaviruses (hrHPV), with a conservative estimate of about 3% -8% of cervical cancers being unrelated to hrHPV infection. Of cervical cancers, more cervical cancer histopathologic types are squamous cancers, a small percentage of which are about 20% adenocarcinoma, while about 15% -20% of adenocarcinomas often appear hrHPV negative. Traditional cervical cancer screening, diversion and referral means include visualization, cytology, hrHPV detection, DNA methylation detection, colposcopy and histopathological biopsy (gold standard). The cytological examination cost is relatively low, but the specificity is poor, no reproducible mode for computer-aided judgment of cervical abscission cytology exists at present, and cytological examination result interpretation is still influenced by subjective judgment of cytologists. hrHPV detection is currently the mainstream primary screening modality for cervical cancer, which has higher sensitivity and longer screening time intervals. However, due to the poor specificity, this increases the number of colposcopic referrals and causes excessive panic in women, and may also lead to about 5% of cervical adenocarcinoma missed diagnosis that is not hrHPV dependent, thus requiring the use of appropriate screening, diversion methods. DNA methylation detection is an objective and repeatable molecular detection technology, has extremely high specificity and reasonable sensitivity, and is an effective means for reducing unnecessary referral and shunt management of colposcopes. At present, a plurality of gene methylation detection technologies exist, and the BSP-TA clone sequencing technology is a gold standard methodology of the DNA methylation detection technology, but has the defects of complex flow, complicated operation, long time consumption, low flux, high clone sequencing cost and the like, and is not suitable for high-flux screening of large sample size, and the method is more suitable for being used as a verification means of the accuracy of other DNA methylation detection methodologies. Pyrosequencing, WGBS and other sequencing technologies, although with higher flux, have the disadvantages of high sequencing cost, wide coverage range, poor sensitivity, long detection and analysis time consumption and inapplicability to rapid detection. Methylation-specific PCR (MSP) technology can detect DNA regional methylation, but two pairs of primers, namely a methylated primer and an unmethylated primer, are required for detection sites, so that multi-target detection is difficult to realize one-tube multiplexing, and an electrophoresis technology is often required during analysis, so that the operation is complicated, and the real-time detection cannot be realized. Quantitative methylation specific PCR (quantitative Methylation-SPECIFIC PCR, QMSP) is an innovative methylation detection high-sensitivity quantitative technology based on MSP, and is matched with a fluorescent PCR instrument to realize the purpose of instant detection, the specificity is improved through a TaqMan probe, one-tube multiplex is realized, the detection can be realized only by a methylation primer probe, the technology has relatively high flux and good sensitivity, and the technology is suitable for rapid screening. The kit based on qMSP platforms is used for screening cervical cancer, but has certain defects such as detection of cervical cancer only, incapability of accurately detecting cervical high-level intraepithelial neoplasia, higher use cost due to methylation pretreatment by using a relatively expensive enzyme method, incomplete coverage of markers, poor inclusion, incapability of realizing detection of types such as non-HPV-dependent cervical cancer, adenocarcinoma and the like, poor suitability, incapability of adapting to cervical exfoliated cells preserved by different cell preservation solutions on the market, larger sample consumption (1 mL-2 mL), incapability of realizing multipurpose detection of one tube of samples, poor methylation detection sensitivity and the like. CN112048561A discloses a composition and a kit for cervical high-level lesions and early detection of cervical cancer, and specifically comprises the following steps of carrying out joint detection on three genes of FAM19A4, JAM3 and PAX1 as core markers, and mainly solving the technical problem of nonspecific amplification (false positive) caused by incomplete conversion of bisulfite. The sample types to which they are directed are cervical exfoliate