engleski

Comparison of Different PCR Methods for Papillomavirus Detection

Human papillomaviruses (HPVs) belong to the family Papovaviridae. HPVs are strictly species specific and epitheliotropic ; they infect epithelial cells either of the skin or the anogenital and oropharyngeal mucosa. Until now, 130 HPV types have been identified and fully sequenced. Approximately 40 types infect the anogenital tract and a few types are commonly found in anogenital cancer biopsy specimens, notably cervical cancer. Detection and typing is useful and important for the diagnosis of HPV associated diseases, notably cervical precancerous lesions and cervical cancer. The molecular methods used for HPV testing are based on the method of hybridization, DNA amplification (polymerase chain reaction - PCR) or both. The PCR method is, actually, the most specific and the most sensitive for revealing the presence of otherwise undetectable quantities of HPV DNA. The method allows detection of wide spectrum of HPVs by using general (consensus) primers. The identification of HPV types may be performed either by hybridization with type-specific probes or by PCR using type-specific primers. In this study, 100 DNA samples, isolated from cervical scrapes with abnormal cytology were analyzed for the presence of HPV by PCR. Five pairs of type specific (TS) primers for HPV 6/11, 16, 18, 31 and 33 (Husnjak et al., J Virol Methods, 2000) and 4 sets of consensus primers: MY09-MY11 (Manos et al., Cancer Cells, 1989), L1C1/L1C2-1/L1C2-2 (Yoshikawa et al., Int. J. Cancer, 1990), PGMY09-PGMY11 (Gravitt et al., J. Clin microbial, 2000) and LCR-E7 (Sasagawa et al. 2000) were used. The most widely used consensus primers are MY09-MY11 primers that are degenerated primers, while PGMY09-PGMY11 consists of a set of 5 forward and 13 reverse primers located in the same region of the HPV L1 gene. Both are generating amplicon of approximately 450 bp depending on the HPV type. L1C1/L1C2-1/L1C2-2 primers consist of one forward and two reverse primers also located in the HPV L1 gene upstream of the MY09-MY11 primers and generating amplicon of approximately 250 bp depending on the HPV type. LCR-E7 primers consist of 4 forward degenerated primers located in the LCR region and 4 reverse primers located in the E7 region generating amplicon of 600 to 758 bp depending on the HPV type. There were 50 negative and 50 positive TS PCR samples. Each sample was tested by 4 sets of consensus primers, MY09-MY11, PGMY09-PGMY11, L1C1/L1C2-1/L1C2-2 and LCR-E7. Our preliminary findings indicate that MY09-MY11 and LCR-E7 primer set had similar sensitivity but both are less sensitive than L1C1/L1C2-1/L1C2-2 and PGMY09-PGMY11. In conclusion, in order to detect the wide range of HPV types by PCR in a clinical sample it is necessary to use the most sensitive and specific consensus primers. In clinical practice, beside consensus primer directed PCR for HPV DNA screening, type specific primers directed PCR should be used for detection of the most common oncogenic HPV types in a certain population that is HPV 16 in Europe.

Human papilloma virus (HPV); polymerase chain reaction (PCR)

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