Development of a Broad Spectrum PCR Assay for Papillomaviruses and its Application in Screening Lung Cancer Biopsies

Abstract

A PCR assay was developed to detect known and as yet unidentified papillomaviruses (PVs). For this purpose we analysed the conserved amino acid sequences in the L1 and E1 open reading frames of 45 human and nine animal PVs. Candidate regions for the design of a primer were identified as those having the least number of amino acid and nucleotide sequence variants among the different PVs. These regions in the L10RF have been described previously. We modified the sequences of the backward and the forward primers, as well as the sequence of the oligonucleotide used as the degenerate probe, in order to cover a broader spectrum of PVs. The sensitivity of the assay for the human and animal PVs tested after hybridization with a 32P-labelled degenerate oligonucleotide probe was one genome copy per cell for integrated PV DNA and 10 genome copies per cell for plasmid PV DNA. The only exceptions were human papillomavirus (HPV) type 4, HPV60 and HPV65, for which a lower sensitivity was obtained. This group could be detected only by using additional primers. The assay was used to analyse 85 lung cancer biopsies representing different histological types. Using this system no PV DNA sequences were detected in the biopsies when compared with human placental DNA (a negative control) and PV DNA-positive standards.

Detection and expression of human papillomavirus oncogenes in non-small cell lung cancer

“…However, reports concerning the HPV detection in lung tumours show several discrepancies. Several studies reported the presence of HPV-DNA within tumours with variable frequency (11)(12)(13)(14)(15)(16)(17)(18). HPV16 and 18 genotypes were the most frequently found in lung tumours.…”

Evaluation of a Novel Highly Sensitive, Broad-Spectrum PCR-Reverse Hybridization Assay for Detection and Identification of Beta-Papillomavirus DNA

“…So far, more than 100 different PV genotypes have been identified, of which approximately 48 types have been detected in human cutaneous lesions (12). These include the beta-papillomavirus (beta-PV) genus comprising the human papillomavirus (HPV) types 5,8,9,12,14,15,17,19,20,21,22,23,24,25, 36, 37, 38, 47, 49, 75, 76, 80, and 93 and candidate types 92 (cand92) and cand96. Based on partial sequences, however, probably more than 35 new types have to be added to the 25 known beta-PV types (19).…”

Detection of human papilloma virus (HPV) and K-ras mutations in human lung carcinomas

“…The rate of positivity varied between 4.2% and 31% (19)(20)(21), while in carcinomas with condylomatous changes the rate was higher (42%) (20). In contrast other investigators reported absence of HPV in lung cancer (37,38), this divergence may be due to the differences in the sensitivity and specificity of the methods applied, as well as to epidemio-logical factors. In the current study we confirm the presence of HPV in lung carcinomas, using primers from both the E6 and LI ORFs.…”