Analytic sensitivity of the HPV-type spectrum detected for both dot blot and line blot assays was determined by serial dilution of HPV plasmid or M13 phage clones amplified in a background of 12.5 ng of human cellular DNA from the K562 cell line (ATCC CCL243). HPV types 58, 59, 61, 62, 64, and 67 were provided by T. Matsukura; HPVs 33, 39, 42, 54, 55, 66, 68, and 70 were from G. Orth; HPVs 6, 11, 16, 18, 53, and 57 were from E. M. de Villiers; HPV 52 was from W. Lancaster; HPV 26 was from R. Ostrow; HPV 45 was from K. Shah; and HPV 51 was from S. Silverstein. Clinical HPV types, including MM4 (W13B), MM7 (P291), MM8 (P155), and MM9 (P238A) had been previously cloned as PCR fragments of approximately 450 bp (16
). Additional sensitivities were determined in precharacterized cervical specimens. Sensitivities were virtually identical by both dot and line blot assays, ranging from 10 to 100 genomes per PCR for HPV types 6, 11, 16, 18, 31, 33, 39, 45, 51, 52, 58, 59, 66, and 68 and from ~500 to 1,000 genomes per PCR for HPV types 26, 35, 40, 42, and 53 to 57. Variation in sensitivity among the genotypes reflects the number and position of mismatched bases in the primer-binding region at nondegenerate sites.
The specificity of HPV genotype discrimination was tested by hybridization of 500 ng (determined by gel quantitation) of amplified product to the HPV genotyping strips. Specificity of typing was excellent, with negligible background or cross-reactivity.
To test the utility of the line blot HPV detection method in clinical samples, we analyzed 359 specimens collected in Digene specimen transport medium. Type-specific oligonucleotide probe results obtained by the standard MY09-MY11-HMB01 dot blot hybridization method were compared to those obtained using the MYB09-MYB11-BHMB01 reverse line blot method. Two separate aliquots of each digested STM sample were taken and processed independently at the two participating laboratory sites, where all aliquots were amplified by using the ultrasensitive amplification profile (see Materials and Methods). Thirty-two HPV-positive and 24 HPV-negative samples determined by the ultrasensitive cycle system were randomly chosen for analysis by the short cycle profile, in which the time at each temperature step in the thermal profile was shortened. Samples were amplified separately for dot and line blot detection because of the requirement of unlabeled versus labeled primers in the dot and line blot detection methods, respectively. Investigators performing the two assays were blinded to results until all interpretations were final.
Of the 359 samples evaluated, 30 were excluded because of false signal generation from the ECL substrate (1
), presumably caused by pseudoperoxidases in the sample, thus precluding interpretation of the dot blot results. The results from the remaining 329 samples are presented.
The HPV prevalence in this population was 24.0 and 25.5% by the dot blot and line blot detection methods, respectively. Table represents the overall HPV concordance between the two detection formats. Agreement for HPV-positive results was good, with a kappa statistic of 0.78. Type-specific agreement between the two methods was good, with total concordance ranging from 97 to 100%. Within the HPV-positive samples, multiple HPV types were detected in 10.7 and 8.5% of specimens by the dot blot and line blot detection methods, respectively. A comparison of the results from 56 samples that were amplified and detected with a long and short cycle profile is presented in Table . Only 49 of the 56 samples were included in the final analysis due to false ECL signals on the dot blot. As expected, the agreement between the two amplification profiles reflected the increase in detection of low levels of HPV with the longer, ultrasensitive profile. Only the line blot results for rapid versus ultrasensitive amplification profiles are shown; however, the dot blot results were virtually identical (87.8% agreement, kappa = 0.75 for both line and dot blot). Further analysis of HPV data for both line and dot HPV assay was conducted based on recorded intensities. Signal intensity scores were as follows: 1, strong; 2, medium; 3, weak; 4, very weak; and 0, negative. Stratified analyses by signal intensity revealed that a short versus a long profile resulted in discordance within HPV-positive specimens designated 3, 4, and 0 (i.e., weak or negative) for both the line and dot blot assays.
TABLE 3 Correlation between ultrasensitive and rapid PCRresultsa
Assays of samples with discrepant results were repeated by line blot. Results by line blot were consistent after repeat analysis, with the exception of weak-positive signals, which were inconsistently amplified. To ascertain the possibility of irreproducibility due to sampling error from low concentrations of viral DNA, we added HPV 16 plasmid DNA to a PCR premix for a final concentration of 1.27 × 10−4 fg/μl, the equivalent of a single target per 100-μl reaction mixture. This mixture was aliquotted to 80 PCR tubes and amplified under sensitive amplification profiles. Analysis of the products by strip analysis indicated that only 42 of 80, or 52.5%, were positive for HPV DNA. Human DNA was included at a concentration of 2.5 ng per PCR and was amplified in all 80 reactions.