Identification of multiple genital HPV types and sequence variants by consensus and nested type-specific PCR coupled with cycle sequencing.
Consensus and type-specific HPV primers were employed for PCR and cycle sequencing of genital HPVs in scrapings and colposcopically directed biopsies of the cervix from a cohort of 188 female sex workers. A total of 27 individuals tested positive for a broad spectrum of HPV types, including HPVs 6b, 16, 18, 31, 33, 34, 35, 45, 56 and 58, as well as a new HPV type, with seven individuals displaying dual infections. Good correlation between the results of individually paired samples was observed. A HPV 16 primer biotinylated at the 5' end was also used as a probe, which could successfully detect amplified products of HPV 16 but not other HPV types tested by an automated ELISA detection system. DNA sequence analysis revealed several HPV sequence variants that harbored mutations, especially in the E6 gene, many of which culminated in non-conservative amino acid substitutions in the transforming E6 oncoprotein. Such an approach of coupling PCR with cycle sequencing permits the determination of many known and even novel HPV types associated with varying degrees of risk to cervical carcinogenesis, and enables the identification of HPV sequence variants of putative biological and clinical significance, thus justifying its utility as an adjunct tool to complement cervical cytology and colposcopy. This study also emphasises the need for educational, interventional and behavioral modification to minimise HPV transmission, such as through consistent condom usage among sex workers.
Chow
V T
VT
Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore. micctk@nus.edu.sg
Loh
E
E
Yeo
W M
WM
Tan
S Y
SY
Chan
R
R
D002584:Cervix Uteri / Q000821:virology; D015331:Cohort Studies; D016384:Consensus Sequence; D004279:DNA, Viral / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006367:HeLa Cells; D006801:Humans; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000635:transmission / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D011477:Sex Work; D015229:Sexually Transmitted Diseases, Viral / Q000635:transmission; D014412:Tumor Virus Infections / Q000635:transmission / Q000821:virology*; D002583:Uterine Cervical Neoplasms / Q000821:virology*
D004279:DNA, Viral