Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus

Abstract

Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1, 354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5(+)/6(+) primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.

FIG. 1

Schematic representation of the locations of the different general primer sets (MY 09/11, GP5⁺/6⁺, and SPF) on the HPV genome. The circular HPV DNA genome is represented by a single line, and boxes show the positions of the various early (E) and late (L) genes. Within the L1 region, the positions of the amplification targets as well as the expected amplimer sizes for each of the primer sets are indicated.

FIG. 2

Alignment of 65-bp nucleotide sequences from the L1 regions of a total of 39 HPV genotypes. The target sequences for the SPF primers are boxed and flank the 22-bp interprimer region, as indicated. Positions are according to the HPV-16 sequence PPH16 (GenBank accession no. K02718).

FIG. 3

Outline and representative examples of the INNO-LiPA HPV genotyping assay. The positions of the control line and the 28 specific probes for each of the 25 HPV genotypes are shown at the left. The number above each strip indicates the HPV genotype for which DNA was amplified by SPF primers. The precise interpretation of the hybridization patterns is described in detail in Materials and Methods.