Detection Of Human Papillomavirus In Clinical Samples And Molecular Cloning Of Viral L1 Gene

Human Papillomavirus (HPV) is the major pathogen of cervical cancer in women, accounting for approximately 500,000 deaths worldwide. Due to lack of an efficient and reliable method for HPV detection, information for HPV infection rate and type distribution has been not yet available in our country. In order to attack the problem, we collected a total of 122 cervical cell samples from women visited OBGYN clinics and 22 tissue samples Condylomata acuiminata (CA) in Bejing area. HPV consensus and type-pecific oligo primers were designed for 12 of high-risk HPV types plus 2 low-risk types, and SSP-PCR were conducted to examine all samples for viral DNA. Initial results showed that approximately 59% of samples were positive for HPV when consensus primers were used. When 12 pairs of HPV type-specific primers were used, the detection rate jumped up to 95.1% and 89%, respectively, for 61 samples diagnosticed as abornormal cytology and 55 random clinical samples. HPV6 or 11 was detected in all 22 CA samples, with 100% detection rate. The study found that all 12 high-riks HPV types were detected in 61 samples with abnormal cytology, and the order of frequencies for the 5 most aboundent HPV types was HPV18 (83.6%) > HPV33 (53.0%) > HPV16 (39.3%) > HPV56 (24.6%) > HPV58 (23.0%). Similarly, all 12 high-risk HPV types were found in 55 random clinical samples, with the 5 most frequent types ranked as HPV33 (75.0%) > HPV18 (65.5%) > HPV56 (27.3%) > HPV16 (25.5%) > HPV39 (21.8%). TA-cloning and subsequent sequencing confirmed the identity of the PCR fragment as that 14 sepcific HPV types. The seuqnece data was deposited in GenBank (Accession number DQ003066-DQ003079). The results showed the combined infection by 2 or more HPV types in majority of samples examined. Approximately 30% of 61 cytologically abnormal samples was infected with three different HPV types, and over 13.8% of the samples has at least 4 different HPV types. The most frequent (30.8%) sample was those infected with two HPV types in random clinical samples, although 3, 4, and even 5 different HPV types was also found. A full-length viral DNA encoding L1 capsule protein of HPV33 was sufessfully cloned. In summary, we have established a sensitive and reliable laboratory procedure for HPV detection and viral classification in clinical samples. This research report for the first time that infection by high-risk HPV in women is surprsingly high, and the HPV type profile could be quite different in Chines population when compared with those abroad.