多重实时PCR对宫颈癌及癌前病变HPV-16病毒整合状态的检测

Detection of viral integration of HPV-16 in the specimens of the cervical carcinoma and it's precancerosis by multiplex real-time PCR.

目的探讨检测HPV存在状态的理想方法,了解HPV-16整合在宫颈癌发生发展中的作用及其可能的临床应用价值。方法用多重实时PCR同管定量检测HPV-16的E2和E6基因,根据E2/E6判定HPV-16的存在状态;与Southern杂交检测结果进行比较。检测HPV-16阳性的宫颈鳞癌51例和宫颈上皮内瘤病变49例的石蜡标本。结果(1)多重实时PCR所得HPV-16E2、E6标准曲线的相关性好,扩增效率均高于95%;(2)确定多重实时PCR以E2/E6比值为0.81作为区分游离型和混合型HPV-16的界值;(3)多重实时PCR与Southern杂交检测的符合率为81.5%(Kappa值为0.844,P<0·001);(4)HPV-16整合发生率与不同程度的宫颈病变有关,CINⅠ、CINⅡ、CINⅢ和SCC中HPV-16整合发生率分别为42.9%、76.5%、83.3%和90.2%;(5)Ⅱ+Ⅲ期宫颈癌中整合型HPV-16所占比例(88.0%)显著高于Ⅰ期宫颈癌(33.3%)。结论多重实时定量PCR是检测HPV-16病毒存在状态的可靠方法,有准确、省时、简便快速及高通量的优点。

Objective ：To search for an ideal method of detecting the existent status of HPV-16 for clinic application and to investigate the role of HPV-16 integration in the cervical carcinogenesis and its potential clinical significance. Methods：Multiplex real-time PCR was applied to quantify the ratio of E2 gene copy number to E6 gene copy number（E2/E6） by which the existent status of HPV-16 DNA was decided. Multiplex real-time PCR was compared with Southern blot analysis in order to verify the method. The method was used to investigate the integrated status of HPV-16 in HPV-16 positive paraffin-embedded tissues including 49 cases of CINs and 51 cases of cervical squamous cancer. Result： （ 1 ）There was a linear relationship between the threshold cycle values and the log of the copy number in both E2 and E6 standard curves,and the amplification efficiencies were both above 95% ;（2）The cut-off value of E2/ E6,which was used to distinguish episomal form from mixed form of HPV-16,was 0.81 in the multiplex real-time PCR test; （3）The rate of agreement between multiplex real-time PCR and Southern blot was 81.5% （ the integration was associated with Kappa statistic was 0. 844 ,P 〈0. 001 ） ; （4）The rate of HPV-16 the degree of cervical lesions. Moreover, the rate increased with the progression of cervical disease,42.9% for CIN Ⅰ ,76.5% for CIN Ⅱ,83.3% for CIN Ⅲ and 90.2% for SCC ; （5） The rate of pure integrated HPV-16 in stage Ⅱ ＋ Ⅲ （88.0%） was significantly higher than that in stage Ⅰ （33.3%）. Conclusions： Mutiplex real-time PCR test is a rapid,sensitive and reliable method for detection of the existent state of HPV-16 DNA.