Single-tube real-time nested polymerase chain reaction for detecting humanpapillomavirus DNA

A single-tube real-time nested polymerase chain reaction (PCR) was develope d to detect human Papillomavirus (HPV) DNA in a closed tube system. The oli gonucleotide primers MY09/MY11 and GP5+/GP6+ were included in contiguous re actions, thus eliminating the need to transfer first round PCR product into a second tube. The sensitivity and specificity of the optimized single-tub e nested PCR were comparable with that achieved by two separate reactions o n a conventional thermal block system using serial dilutions derived from p lasmids containing DNA of 20 HPV types, A minimum of 10 copies of HPV types 11 and 16 DNA could be detected by both systems. In clinical samples, HPV types 1A, 2, 3, 5, 6-8, 10, 11, 14, 16, 17, 18, 20, 31, 33, 35, 39, 45, 49, 50, 52-54, 57, 62, 66, 70, CP8304 and LVX82/MM7 could be detected by both PCR methods. A total of 145 samples collected from patients were tested for the presence of HPV DNA with the two PCR systems: 124 (86.1%) of 144 sampl es gave concordant results in both assays. The HPV DNA positive PCR amplico ns were typed and concordant results were obtained in 47 of 67 positive sam ples tested in both amplicons. In samples containing multiple HPV types at least one type was common to both amplicons.