Human papillomavirus genotyping using a modified linear array detection protocol.
A standardized PCR-based linear array genotyping assay has been developed (Roche Molecular Systems) allowing the concurrent typing of 37 human papillomavirus (HPV) types. A component of this assay, the PCR amplicon detection protocol, requires the use of a shaking water-bath incubator for amplicon/probe hybridization and subsequent stringent washing, which many laboratories may find cumbersome or simply not possess. In this study, the utility of a dry-air incubator in substitution of the recommended shaking water-bath for use in this assay was confirmed. DNA was extracted by AmpliLute method from 150 PreservCyt specimens collected from patients undergoing ablation treatment for histologically confirmed cervical abnormality. The DNA was amplified by PCR and amplicons detected by both standard and modified protocols, varying only by the method of incubation for DNA hybridization and stringent washing. Identical HPV-type profiles were generated in 149/150 (99.3%) of the specimens tested by both protocols, representing a near perfect level of concordance (kappa = 0.98). It was proposed that this modification would markedly simplify the detection process, reduce setup time and eliminate the potential for water spillover, thereby allowing more laboratories to adopt this method subsequent to in-house validation.
Department of Microbiology, The Royal Women's Hospital, Carlton, Vic., Australia. matthew.stevens@mcri.edu.au
Matthew P
MP
Stevens
Suzanne M
SM
Garland
Sepehr N
SN
Tabrizi
D002584:Cervix Uteri / Q000821:virology; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics*; D005260:Female; D005838:Genotype; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*
D004279:DNA, Viral