Detection and typing of human papillomavirus by e6 nested multiplex PCR

Abstract

A nested multiplex PCR (NMPCR) assay that combines degenerate E6/E7 consensus primers and type-specific primers was evaluated for the detection and typing of human papillomavirus (HPV) genotypes 6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66, and 68 using HPV DNA-containing plasmids and cervical scrapes (n = 1,525). The performance of the NMPCR assay relative to that of conventional PCR with MY09-MY11 and GP5+-GP6+ primers, and nested PCR with these two primer sets (MY/GP) was evaluated in 495 cervical scrapes with corresponding histologic and cytologic findings. HPV prevalence rates determined with the NMPCR assay were 34.7% (102 of 294) in the absence of cervical intraepithelial neoplasia (CIN 0), 94.2% (113 of 120) in the presence of mild or moderate dysplasia (CIN I/II), and 97.8% (44 of 45) in the presence of severe dysplasia (CIN III). The combination of all four HPV detection methods applied in the study was taken as "gold standard": in all three morphological subgroups the NMPCR assay had significantly (P < 0.0001) higher sensitivities than the MY09-MY11 and GP5+-GP6+ assays and sensitivities comparable or equal to those of the MY/GP assay. All 18 HPV genotypes investigated were detected among the clinical samples. The ratio of high- to low-risk HPV genotypes increased from 4:1 (80 of 103) in CIN 0 to 19:1 (149 of 157) in CIN I to III. Multiple infections were detected in 47.9% (124 of 259) of the patients. In conclusion, the novel NMPCR method is a sensitive and useful tool for HPV DNA detection, especially when exact HPV genotyping and the identification of multiple HPV infections are required.

FIG. 1.

HPV detection and typing by GP-E6/E7 NMPCR. Diagram of PCR amplicon positions relative to the HPV-16 genome. Type-specific nested PCR primers were arranged in four cocktails.

FIG. 2.

Amplification of HPV DNA-containing plasmids (100 pg of plasmid DNA; 10⁷ viral copies) with consensus primers GP-E6-3F and GP-E7-5B/6B. The PCR products generated with GP-E6/E7 primers from HPV-containing plasmids and from a clinical sample (HPV-51) range from 602 to 666 bp in size. HPV genotypes are indicated at the top. M, length marker ΦX 174 phage DNA, digested by HaeIII.

FIG. 3.

Type-specific identification of HPV DNA. Eighteen primer pairs for type-specific HPV detection by nested PCR were arranged in four multiplex cocktails. The ability of each primer pair to detect HPV DNA is demonstrated by amplification of the corresponding HPV type from a single plasmid [lanes (+)] and from a cocktail containing all 18 HPV genotypes addressed in the study (lanes +). The specificity of each primer pair was demonstrated by the absence of unspecific amplification products when the HPV plasmid or HPV DNA belonging to the primer tested was missing from the cocktail (lanes −). In all experiments 100 pg of plasmid DNA (10⁷ viral copies) was amplified.

FIG. 4.

Sensitivity of MY09-MY11 and GP5+-GP6+ nested PCR (MY/GP) and NMPCR. A 10-fold dilution series of HPV DNA-containing plasmids of genotypes 6, 35, 52, and 66 was amplified by both methods. Lanes 1 to 7 represent PCR products of 10 pg to 10 ag (10⁶ to 1 viral copy). MY09-MY11 and GP5+-GP6+ nested PCR is 10 times more sensitive for HPV-6, while the NMPCR method is 10 times more sensitive for HPV-35 and HPV-66 and nearly 100 times more sensitive for HPV-52. M, length marker ΦX 174 phage DNA, digested by HaeIII.

FIG. 5.

HPV detection and typing by GP-E6/E7 NMPCR. The results of 13 samples with multiple infections are shown (lanes 1 to 13). The individual lanes in each of the four gels represent amplification products from the same sample. The genotypes identified per primer cocktail are indicated below each gel. Not only the specific detection of the various HPV genotypes as determined by the amplification of control DNA (lanes 4 and 5 on the right), but also the ability of the assay to detect infections with multiple HPV genotypes within each multiplex primer cocktail is demonstrated (lanes 2 and 4 to 12). One hundred femtograms of HPV DNA-containing plasmids was amplified under the same conditions and served as positive controls. M, length marker ΦX 174 phage DNA, digested by HaeIII.