Improvement of PCR detection of HPV-DNA using enhanced chemiluminescence system and dot hybridization.
Infection with human papillomavirus (HPV) is an important etiological factor in the development of cervical cancer; detection of the viral genome is of prognostic importance, particularly for preneoplastic lesions. Nowadays, polymerase chain reaction (PCR) technique is a method of choice for HPV detection because of its sensitivity. However, providing a reliable diagnostic test, both sensitivity and specificity of the test should be evaluated. In the present study, purified plasmid HPV-16 DNA and HeLa-DNA, containing HPV-18 DNA, were amplified by PCR using L1 consensus primers specific for HPV. The amplified product was then analysed by gel electrophoresis (GE) and dot hybridization (DH). The generic oligonucleotide probes (GPs) labelled with Enhanced Chemiluminescence 3' labelling kit (ECL) were used in DH. The sensitivity of PCR reaction after determining by GE was 1 copy per cell for purified plasmid HPV-16 DNA and 20-80 copies per cell for HeLa-DNA while determining by DH was only 0.1 and 2-8 copies per cell, respectively. Thus, the detection of amplified product by DH using enhanced chemiluminescence system improved not only the specificity but also the sensitivity of HPV detection at least 10-fold.
Poonnaniti
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A
Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Bhattarakosol
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P
D004279:DNA, Viral / Q000302:isolation & purification*; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004279:DNA, Viral