Genotyping human papillomaviruses: Development and evaluation of a comprehensive DNA microarray

Abstract

GoalsTo define the analytical and clinical performance of a human papillomavirus (HPV) custom-designed microarray targeting the HPV L1 gene for viral genotyping. MethodsMicroarray probes were designed by cataloging the genome sequence of all 120 known HPV types to generate tiling probes using eArray® software against the unique L1 capsid gene segments targeted by MY09/11 and FAP59/64 primers. The microarray (1 slide×8 arrays×60K features) synthesized in situ by inkjet printing was tested using synthetic type-specific HPV DNA and existing HPV DNA from cervical cytology. The synthetic HPV L1 segments (genotypes 6, 11, 16, 18, 31, 33, 35, 45, 53, 58, 66, 73, 83) were manufactured from sequences stored in the NCBI taxonomy database. Using the hybridization patterns of the synthetic HPV DNA as the Support Vector Machine classifier, HPV DNA from patient samples were genotyped and compared to antecedent DNA sequencing/BLAST® results for concordance. Results16 cytology-derived HPV DNA samples and 13 synthetic type-specific HPV DNA samples were tested singly, in duplicate, or in combination on 40 arrays. The synthetic HPV DNA hybridization patterns were found to be uniquely distinctive to serve well as a classifier of unknown HPV-containing specimens. For the 16HPV DNA+ samples classified, 15 were concordant with DNA sequencing results. In 6/16 (38%) samples, the microarray hybridization pattern revealed ≥2 concurrent HPV infections. ConclusionThe novel “HPV Array” was sensitive and specific for detecting single and multiple infections. This proof-of-principle project demonstrated the accuracy and advantages of microarray technology for HPV genotyping.