Evaluation of a novel highly sensitive, broad-spectrum PCR-reverse hybridization assay for detection and identification of beta-papillomavirus DNA.
Human papillomavirus can be detected by amplification of viral DNA. A novel one-step PCR (PM-PCR) was evaluated for amplification of a 117-bp fragment from the E1 region. It permitted ultrasensitive detection of all 25 known human papillomavirus genotypes from the beta-papillomavirus genus. The intra- and intertypic sequence variations of the 77-bp interprimer region were studied. Genotype-specific probes as well as general probes were selected for the 25 established beta-papillomavirus types, and a reverse hybridization assay (RHA) was developed (PM-PCR RHA method). The analytical sensitivity of the PM-PCR RHA method was 10 to 100 viral genomes. The one-step PM-PCR turned out to be more sensitive than the previously described nested MaHa-PCR for beta-papillomavirus detection. The PM-PCR RHA method was able to detect and identify beta-papillomavirus types in frozen patient material as well as in poorly amplifiable material such as formalin-fixed, paraffin-embedded skin biopsy specimens. Inter- and intralaboratory variability experiments showed that the reproducibility of the assay was very high. In conclusion, the one-step PM-PCR together with the RHA allows extremely sensitive, specific, and reproducible detection of beta-papillomavirus DNA as well as reliable identification of beta-papillomavirus genotypes in both fresh and paraffin-embedded patient material.
de Koning
Maurits
M
Delft Diagnostic Laboratory, Fonteijnenburghlaan 5, 2275 CX Voorburg, The Netherlands.
Quint
Wim
W
Struijk
Linda
L
Kleter
Bernhard
B
Wanningen
Patrick
P
van Doorn
Leen-Jan
LJ
Weissenborn
Sönke Jan
SJ
Feltkamp
Mariet
M
ter Schegget
Jan
J