HPV Typing Detection And Study Of Carcinogenesis Of HPV Methylation By Pyrosequencing

Partâ… Hr-HPV typing detection in remaining cells of TCT by pyrosequencingObjective:To evaluate the value of pyrosequencing in typing detection of Hr-HPV infection. To analysis the accuracy of this method, the effectivity of detection high-level cervical lesion, and the feasibility of clinical test and large-scale screening by pyrosequcing. To gain the rate and types of Hr-HPV infection in the patients of colposcopy clinic.Content:200 patients of colposcopy clinic was the object of the study. TCT, HC-â…¡test, and Hr-HPV typing test by pyrosequencing were performed in all of the patients. Analysis the value of identifying Hr-HPV by pyrosequencing, and the consistency of pyrosequencing and HC-â…¡in detecting Hr-HPV. Evaluate the effectivity of detection high-level cervical lesion by this new method.Method:12 types of Hr-HPV were tested by prosequencing. DNA was extrated from remaining cells of TCT, then PCR were performed with the general primers of HPV. Hr-HPV typing was tested by pyrosequencing in positive PCR amplicators. In order to save testing time, every four sequencing primers were put into one pool of sequencing. Only 10 minutes were spent in sequencing. Analysis the consistency of pyrosequencing and HC-â…¡which is the only approved HPV clinical test method of FDA. Evaluate the effectivity of pyrosequencing in cervical cancer screening. Purificate, clone, and sequencing the DNA which was positive in PCR, but negtive in pyrosequencing in order to confirm the type of HPV.Result:98 cases of Hr-HPV infection were detected by pyrosequencing.88 were sigle infection, and 10 were double co-infection.9 types of Hr-HPV were identified, including HPV16, HPV18, HPV31, HPV33, HPV39, HPV45, HPV52, HPV58, and HPV59. HPV16 was the most popular Hr-HPV type, it accounted for 69.4%. HPV58 was the second one, it accounted for 16.3%. Hr-HPV infection detecting by pyrosequencing was positive corelated with cervical lesions(Kendall=0.704, P<0.001). The sensitivity and specificity of identifying high-level cervical lesion by this method was 95% and 65.1%, respectively. The area under ROC curve was 0.80. Pyrosequencing was well consistent with HC-â…¡(Kappa=0.791, P<0.001). Conclusion:Hr-HPV detection by pyrosequencing is well cosistent with HC-â…¡. Furthermore, typing detection of Hr-HPV can be performed by pyrosequencing. CINâ…¡and more serve cervial lesion can be detected by pyrosequencing.Partâ…¡Quantificationally and located measure the mythelation rate of CpG of HPV 16 and evaluate the relationship between methylation and it's pathogenicity.Objective:Quantificationally and located measure the mythelation rate of 21 cytosine guanine dinucleotide(CpG) sites in the L1 3'and long control region (LCR) of HPV 16 DNA in asymptomatic patients, cervical intraepithelial neoplasia(CIN) patients, and cervical cancer patients. Analysis the relationship between HPV16 methylation and it's pathogenicity.Content:Asymptomatic, CIN, and cervical cancer patients with HPV16 infection were involved in the second part of study.30 patients were included in each groups. Quantificationally measure the mythelation rate of 21 CpG sites in the L1 3'and long control region (LCR) of HPV 16 DNA. Analysis the relationship between HPV16 methylation and it's pathogenicity.Method:Chose 30 HPV positive cases in each group. Firstly, extract DNA from the remainding cells of TCT specimen and bisulfite treatment DNA, then amplificate the 3'region of L1 and LCR, test the mythelation rate of 21 CpG sites of HPV 16 DNA in three groups. Analysis the relationship between methylation rate of each CpG sites and pathogenicity of HPV16.Result:All of the 5 CpG sited in promoter of E6/E7 were hypermethylation in asymptomatic group, hypomethylation in cervical group, middle-methylation in CIN group. The difference was significant. The CpG site in 7032,7019,7136 of L1 3'region was different methylated in 3 groups. Hypermethylation was found in cancer group, hypomethylation was found in asymptomatic group in 7019 and 7032. In 7136, the highest methylation was detected in CIN, lowest in asymptomatic, middle in cancer.Conclusion:The methylation status of CpG sites in L1 3'region and promoter of E6/E7 was significant different in three groups, and it's the reason of virus DNA integration, oncogene expression and cancer induction. The methylation status of HPV 16 is likely to be the biomarker of identifying recovery and pathogenicity HPV16 infection, anticipating CIN and cervical cancer, and offer a new target of therapy in the future.