Fast Multiplex polymerase chain reaction on boiled clinical samples for rapid viral diagnosis

An assessment of optimal conditions for rapid simultaneous amplification of multiple human papillomavirus (HPV) sequences has been made using Thermus aquaticus DNA polymerase. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of well characterized cell lines. In our hands, Fast Multiplex PCR (FM-PCR), the technique of running multiple PCR reactions simultaneously with minimum incubation time at each temperature, was highly sensitive (amplification factor = 5 x 10(9) after 50 cycles), specific (100%) and reproducible (100%) for several microbiological applications. Diagnosis was generally obtained in less than 5 h after sampling. The results show that, after optimization of assay conditions, efficiency and specificity of Multiplex PCR depends exclusively on the primers design and concentration of the primers.