Evaluation of a novel highly sensitive, broad-spectrum PCR-reverse hybridization assay for detection and identification of beta-papillomavirus DNA

Abstract

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.

FIG. 1.

Nucleotide sequence alignment of the target region for the PM-PCR for 25 beta-PV types. The complete 117-bp product is designated region E and is located between nucleotides 2644 and 2760. The sequence numbering is relative to the HPV5 sequence. Target regions A and D for the forward and reverse primers, respectively, are shown by boxes. The 77-bp area between the primers is designated region C. Region B is the target for the universal probes. The nucleotides identical to the top sequence are indicated by dots.

FIG. 2.

Outline of the PM-PCR RHA method and typical patterns arising upon analysis of PM-PCR-derived amplimers. The top line is the conjugate control, which serves as the positive control for the enzymatic coloring reaction. The other lines are indicated by the probe names, beginning with the mixture of general probes (“Universal”). The remaining lines represent genotype-specific probes, except for probes HPV8 I, HPV8 II, HPV21, and cHPV21, which are used in pattern recognition of genotypes. The tested amplimers were obtained by performing the PM-PCR on plasmid clones of 23 beta-PV genotypes and 2 beta candPV genotypes (genotyping results representing HPV types 5, 8, 9, 12, 14, 15, 17, 19 to 25, 36 to 38, 47, 49, 75, 76, 80, cand92, 93, and cand96 are shown from, respectively, strips 1 to 25).

FIG. 3.

(A) Typical example of the sensitivity range of the PM-PCR RHA method as demonstrated by input of an HPV5 serial plasmid dilution in a background of human genomic DNA (50 ng). Ten microliters of PM-PCR product was analyzed by electrophoresis. The PM-PCR mix control contained water (lane 2). DNA input in the PCR ranged from 10,000 (lane 3) to 1 (lane 7) copies of HPV5 plasmid DNA, with each lane starting from lane 3 representing a 10-fold dilution. Lane 1 contains a 100-bp DNA marker. (B) RHA results of the samples from panel A. The top line is a positive control containing biotinylated DNA, the second probe line is for general beta-PV detection, and the third probe line has the HPV5 type-specific probe.