宫颈非典型增生组织中HPV16－DNA的定量PCR检测

DETECTION OF HUMAN PAPILLOMAVIRUS TYPE 16 DNA BY QUANTITATIVE PCR IN CERVICAL DYSPLASIA TISSUES

①目的建立定量聚合酶链反应（ＰＣＲ）方法，检测宫颈组织中人乳头瘤病毒１６型（ＨＰＶ１６）的基因含量。②方法通过重叠延伸ＰＣＲ构建一含ＥｃｏＲＩ酶切位点的内参照模板，用竞争性ＰＣＲ（ＣＰＣＲ）检测６例ＨＰＶ１６阳性宫颈非典型增生组织标本中ＨＰＶ１６Ｅ６基因的拷贝数。③结果６例阳性标本ＨＰＶ１６Ｅ６基因的拷贝数为每毫克ＤＮＡ中含１．９９×１０５～２．５０×１０７．④结论用ＣＰＣＲ检测宫颈组织中ＨＰＶ１６Ｅ６基因的含量是可行的，进一步比较不同宫颈疾患组织中ＨＰＶ１６Ｅ６基因的含量，可以从分子水平探讨ＨＰＶ１６的致癌机理。

Objective To set up a quantitative polymerase chain reaction method for detecting human papillomavirus type 16 DNA(HPV16) in cervical tissues. Method A HPV16 DNA internal standard template with EcoRI restriction site was prepared by using a PCR technique and site directed mutagenesis by overlap extension. The number of copies of HPV16E 6, DNA in six positive samples of cervical dysplasia was detected by competitive PCR. Result Six out of 14 cervical dysplasia samples were positive. The number of HPV16 DNA copies in the six samples were 1.99×10 5～2.50×10 7 copies/μg DNA. Conclusion the results showed that it was possible to detect the quantities of HPV16 E 6, DNA in cervical tissues by competitive PCR. This technique could be applied to different cervical diseases and help to find the carcinogenesis of HPV16 at molecular level.