Polymerase chain reaction for fast, nonradioactive detection of high- and low-risk papillomavirus types in routine cervical specimens and in biopsies

Abstract

A polymerase chain reaction (PCR) procedure capable of amplifying specific DNA sequences by up to a millionfold was developed for detection of infection by human papillomaviruses (HPVs) of low (HPV6, HPV11) or high (HPV16, HPV18, HPV33) oncogenic potential. For high-risk HPVs the region chosen was within the E6 open reading frame, which can become integrated into genomic DNA. A region corresponding to this was chosen for low-risk HPVs. After repeated cycles of specific oligonucleotide-primed extension of viral DNA with Klenow or thermophilic DNA polymerase, the type of HPV present was then determined on the basis of the size of the ethidium-bromide-stained band visible after polyacrylamide gel electrophoresis: for HPV6 or 11 the band was approximately 120 bp, for HPV 16 or 33 it was approximately 200 bp, and for HPV18 it was approximately 100 bp. Specific hybridization to the relevant band was seen using radioactive or nonradioactive (alkaline-phosphatase-linked) target oligonucleotide probes. Using the PCR method, we have determined, within as little as a few hours, the infection status of a variety of clinical specimens, including cervical scrapes and lavages, anal scrapes, and anogenital biopsies. The PCR steps can be automated, adding to the potential of PCR for widespread use in the detection of HPV, which is becoming increasingly popular in cervical screening.