DETECTION OF HUMAN PAPILLOMAVIRUS IN VULVAR CARCINOMA USING SEMI-NESTED PCR AND RESTRICTION ENZYME TYPING - A RAPID AND SENSITIVE TECHNIQUE

Aims-To develop a highly sensitive technique for the reliable detectio n and typing of human papillomavirus (HPV) DNA in clinical tissue. Met hods-A two step, semi-nested PCR was used with primers spanning the L1 region of the NPV genome and capable of detecting HPV DNA of all know n HPV types. The clinical samples were typed by digestion of the 412 b ase pair PCR product with Rsa I, generating unique fragments for each HPV type. Thirteen samples were screened by this method, including nin e vulval carcinoma samples and four wart samples from the penis and vu lva. Results-Experiments using DNA extracted from HPV DNA positive cel l lines-that is, CaSki (HPV type 16) and HeLa (HPV type 18) establishe d that the technique could detect as few as 50 HPV copies and that the predicted Rsa I fragments from HPV types 16 and 18 were generated. Th e predicted 412 base pair fragment was observed for all 13 clinical sa mples subjected to semi-nested PCR. Rsa I digestion of the product of the second round of PCR permitted the positive identification of the H PV type in most cases. Conclusions-This technique provides an effectiv e and rapid means of detecting HPV DNA, in most cases providing the HP V type. High risk HPV types were always detected in the nine vulval ca rcinoma samples analysed. The amount of tissue available from the biop sy specimens was small, confirming the sensitivity of the method.