Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types

The importance of assays for the detection and typing of human papillomaviruses (HPVs) in clinical and epidemiological studies has been well demonstrated. Several accurate methods for HPV detection and typing have been developed. However, comparative studies showed that several assays have different sensitivities for the detection of specific HPV types, particularly in the case of multiple infections. Here, we describe a novel one-shot method for the detection and typing of 19 mucosal high-risk (HR) HPV types (types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82). This assay combines two different techniques: multiplex PCR with HPV type-specific primers for amplification of viral DNA and array primer extension (APEX) for typing. This novel method has been validated with artificial mixtures of HPV DNAs and clinical samples that were already analyzed for the presence of mucosal HPV types by a different consensus PCR method, i.e., GP5+/GP6+. Our data showed a very good agreement between the results from the multiplex PCR/APEX assay and those from the GP5+/GP6+ PCR (overall rates of HPV positivity, 63.0 and 60.9%, respectively). Whereas the GP5+/GP6+ PCR was slightly more sensitive for the detection of HPV type 16 (HPV-16), multiplex PCR-APEX found a higher number of infections with HPV-33, HPV-53, and multiple HPV types. These favorable features and the high-throughput potential make our present novel assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of mucosal HR HPV types in cervical specimens.