人乳头瘤病毒整合检测方法的建立

Establish a method for detecting HPV integrity

目的研究受感染宫颈上皮细胞中人乳头瘤病毒整合的检测方法与应用。方法构建串联单拷贝基因重组质粒,用于对经反向斑点杂交法基因检测为16型人乳头瘤病毒感染的宫颈脱落细胞样本进行病毒的早基因分析,通过串联单拷贝基因标准化方法,对采用荧光定量PCR技术分析得到的样本中HBB、病毒早基因E2与E6的检测数据分析,计算HPV病毒整合参数与平均病毒拷贝数,推断E2基因完整性及病毒整合发生、病毒拷贝数与已知的病变进程的相关性。结果构建的重组质粒可有效用于标准化同批次荧光定量PCR检测E2基因、E6基因和HBB基因的数据。37例16型人乳头瘤病毒阳性宫颈细胞样本中,15例样本结果为E2基因已破坏,包括10例高度病变组样本及5例正常组样本。不同组E2基因完整性差异有统计学意义(P<0.05)。E2基因被破坏的样本平均病毒拷贝数比E2基因完整样本显著减少(P<0.05)。结论本研究建立的串联单拷贝基因标准化法用于检测16型HPV感染的宫颈细胞样本中病毒整合导致的E2基因破坏有效可行。

Objective To establish a method to detect viral integrity of human papillomavirus in women cervical HPV infection. Methods We amplified E6/E7 gene and E2 gene of HPV16,then inserted them into a plasmid containing single copy HBB gene. HPV16 infected cervical epithelium samples were screened out by genotyping with RDB of flow-through hybridization assay.Fluo-rescence quantitive PCR data of HBB,viral E2 gene and viral E6 gene of all samples were standardized by compared with respective parameters of the plasmid.The ratio IHPV and CHPV were calculated to find out E2 gene disruption and viral copies per cell in the cer-vical samples,respectively.Results The plasmid constructed for standardization was proved effective to make the FQ-PCR data of E2 gene,E6 gene and HBB gene comparable.Thirty-seven HPV16 positive cervical epithelium samples included 22 cases from women whose TCT were normal,and 15 cases from women who confirmed HIL/CIN 2-3 or above through colposcopic examina-tion plus biopsy.Fifteen samples were detected E2 gene disruption,including 10 HIL/CIN 2-3 or above samples and 5 TCT normal samples.E2 gene integrity in different groups were statistically significant different（P 〈0.05）.The average viral copies per cell dis-played a significant decline along with E2 gene disruption（P 〈0.05）.Conclusion The tandem single copy gene plasmid standard-ized methord for the detection of E2 gene disruption caused by viral integration in HPV16 infected cervical cells is feasible and effec-tive.