Real-time duplex PCR for simultaneous HPV 16 and HPV 18 DNA quantitation

HPV 16 and HPV 18 are responsible for more than 75% of cervical cancers and high HPV 16 loads are associated with both prevalent and incident lesions. The objective of the present study was to develop a method allowing the detection and quantitation of HPV 16 and 18 DNA to improve future strategies for cervical cancer screening. A duplex real-time PCR allowing the simultaneous quantitation of both HPV 16 and HPV 18 was carried out. Mixes of HPV 16 and HPV 18 whole genome plasmids were prepared to test a wide range of viral DNA concentrations. The values obtained for each mix of plasmids with the simplex and the duplex PCR were very close to the theoretical values except when a HPV type represented only 1:1000 genome equivalent or lower than the concurrent type. Cervical samples harboring HPV 16, HPV 18 or both types were tested by comparing the results with simplex and duplex real-time PCR assays. HPV 16 and HPV 18 genome titers were similar with the two assays. In conclusion, the real-time duplex PCR proved to be robust for HPV 16 and HPV 18 DNA quantitation.