SIMULTANEOUS POLYMERASE CHAIN-REACTION DETECTION AND RESTRICTION TYPING FOR THE DIAGNOSIS OF HUMAN GENITAL PAPILLOMAVIRUS INFECTION

A polymerase chain reaction method has been developed which allows the simultaneous detection of the majority of clinically relevant HPV typ es. Degenerate HPV-specific primers direct the one-step amplification of a DNA region spanning E1 and E7 genes. This enables an immediate di stinction between the two groups of papillomaviruses, characterized by high or low oncogenic potential, simply from the size of amplified DN A, The PCR product can be subjected to a second round of amplification with internal primers, which are specific for 7 high-risk HPV types, HPV-16, -18, -31, -33, -35, -45 and -58. Precise identification of one -step or two-step amplified DNA is done by endonuclease digestion with one or two enzymes. The detection sensitivity, which has been assesse d using cloned HPV genomes and HeLa and CaSki cell lines, varies from a few tens to a few hundreds of viral genome equivalents. The accuracy of the method has been confirmed by examining cervical scrapings of 4 4 patients.