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Application of a nested multiplex polymerase chain reaction method in the typing of human papillomavirus

Abstract:

Introduction: there are a large number of techniques, both commercial and in house, for detection of human papillomavirus infection. To detect multiple infections, robust techniques are required which provide high sensitivity and specificity. Objectives: implement a nested multiplex polymerase chain reaction protocol for detection and typing of human papillomavirus in anogenital samples from Ecuadorian men. Methods: a study was conducted of 29 samples obtained by anogenital brushing performed Ecuadorian men from two regions in the country (25 samples from the penis and 4 from the anal mucosa of patients with anal condylomata). The protocol was applied using primers aimed at conserved regions of viral genes E6/E7 for an initial polymerase chain reaction. For the second polymerase chain reaction, nested and multiplex, specific primers were grouped in three cocktails of four genotypes, making it possible to discriminate high from low oncogenic risk genotypes, based on the size of the amplified product. Results: 59% of the samples were positive (17/29) with a predominance of the following genotypes: human papillomavirus 43 (31%), 16 (28%), 6/11 (24%), 42 (24%), 58 (14%) and 52 (10%). A higher percentage of samples were found to be infected by a single genotype (41%; 7/17), followed by multiple infection by more than three genotypes (29%; 5/17), two genotypes (18%; 3/17) and three genotypes (12%; 2/17). Conclusions: the protocol applied is specific for the human papillomaviruses analyzed. Use of cocktails of primers with several genotypes makes it possible to discriminate the latter, demonstrating that the technique applied is useful for diagnosing 12 different human papillomavirus genotypes.