Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method

Abstract

Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or GP5(+)/6(+)) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis, genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle. Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albumin-conjugated oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancer-associated, genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of beta-globin probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al., p. 132-152, in C. S. Herrington and J. O. D. McGee (ed.), Diagnostic Molecular Pathology: a Practical Approach, (1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics, Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92% concordance for HPV positivity (kappa = 0.78, P < 0.001). Nearly all of the discrepant HPV-positive samples resulted from weak signals and can be attributed to sampling error from specimens with low concentrations (<1 copy/microliter) of HPV DNA. The primary advantage of the strip-based detection system is the ability to rapidly genotype HPVs present in genital samples with high sensitivity and specificity, minimizing the likelihood of misclassification.

FIG. 1

HPV genotyping of PCR product by reverse line blot method. Schematic of the reverse line blot genotyping assay from L1 consensus primer-generated PCR products. The drawing represents the detection of a hypothetical mixed infection of HPV 16, 31, and 11.

FIG. 2

Probe layout of the HPV genotyping strip. (a) HPV genotyping strips (n = 28) hybridized with the HPV L1 consensus PCR product generated from the HPV targets indicated to the right. Fifty microliters of PCR product generated from amplification of 10⁶ HPV plasmid targets (with the exception of HPV 51 and 68, which were amplified with 10³ plasmid targets) in a background of human cellular DNA (12.5 ng/PCR) was hybridized to the HPV genotyping strips and detected by the previously described reverse line blot method. (b) Line blot genotyping hybridization results for 10 clinical specimens in the previously described study. Fifty microliters of denatured PCR product was hybridized to each strip. The genotyping results for the specimens are as follows: no. 333, HPV negative; no. 334, HPV negative; no. 352, HPV 16, 26, and MM8; no. 353, HPV 16; no. 354, HPV 16, 51, and 66; no. 355, HPV negative; no. 357, HPV 39; no. 359, HPV MM7; no. 361, HPV 16 and 52; and no. 373, HPV 18, 56, and 58.