The PGMY L1 consensus primer pair combined with the line blot assay allows the detection of 27 genital human papillomavirus (HPV) genotypes. We conducted an intralaboratory and interlaboratory agreement study to assess the accuracy and reproducibility of PCR for HPV DNA detection and typing using the PGMY primers and typing amplicons with the line blot (PGMY- Human papillomavirus (HPV) infection is a strong independent predictor for squamous intraepithelial lesions (SIL) and invasive cancer of the uterine cervix (10, 34). Women with persistent HPV infection are at highest risk for development of cervical SIL or evolution from low-grade SIL to higher-grade disease of the uterine cervix (8,24). The causal association between HPV and cervical cancer was demonstrated by epidemiological investigations that used PCR, the most sensitive tool for HPV detection and typing, in cells collected from the uterine cervix (5).LBBecause of the genetic polymorphism of HPV, consensus PCR assays have been utilized to amplify in one reaction the majority of known, as well as novel, anogenital HPV genotypes. The most widely used primer sets, the MY09/MY11/ HMB01, GP5ϩ/GP6ϩ, PGMY09/PGMY11, and SPF1/SPF2 consensus primers, target conserved sequences in the HPV L1 gene (1,7,12,15,18,20,23). To render the consensus PCR assays more feasible for large-scale testing and clinical application, convenient assays for the detection and typing of HPV have been developed for all primer sets. HPV amplicons generated by PGMY primers can be easily detected and typed by a nonisotopic reverse hybridization assay, the line blot (LB) assay (4, 13). Recently, a colorimetric microtiter plate-based enzyme immunoassay was also reported for screening of the broad spectrum of HPV amplified by the PGMY primers using a generic probe mix (21).The proficiency of microbiology laboratory testing is generally monitored by proficiency-testing programs that also allow the determination of test variability between laboratories (32). Implementation of proficiency testing panels is essential for unregulated molecular diagnostic tests. Proficiency panels have been developed for the molecular diagnosis of several infectious agents (25,32) and in research settings to monitor the performance of molecular virology laboratories (17). Thus far, no proficiency panel has been constructed and made available to the general research community for HPV testing. The validity of HPV DNA detection and typing with PCR assays has not been thoroughly assessed.Epidemiological studies and vaccine clinical trials require the reliable and reproducible identification of genital HPV infection. Several studies have evaluated the intermethod variation of HPV DNA detection (2,11,14,19,22,26,28,30,33). However, few studies have evaluated the intralaboratory and interlaboratory reproducibility of L1 consensus PCR assays although these assays have been widely used (6,16,19). The latter studies were conducted with in-house reagents and protocols. Although the consensus L1 PCR assays are not commercially ...
We assessed the value of a new digoxigenin (DIG)-labeled generic probe mix in a PCR-enzyme-linked immunosorbent assay format to screen for the presence of human papillomavirus (HPV) DNA amplified from clinical specimens. After screening with this new generic assay is performed, HPV DNA-positive samples can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplified via PCR using biotin-labeled consensus primers directed to the L1 gene. HPV amplicons were captured on a streptavidin-coated microwell plate (MWP) and detected with a DIG-labeled HPV generic probe mix consisting of nested L1 fragments from types 11, 16, 18, and 51. Coamplification and detection of human DNA with biotinylated -globin primers served as a control for both sample adequacy and PCR amplification. All specimens were genotyped using a reverse line blot assay (13). Results for the generic assay using MWPs and a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were compared with results from a previous analysis using dot blots with a radiolabeled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance in genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positives using the radiolabeled generic probe technique, suggesting slightly improved sensitivity. Only one sample failed to test positive with the DIG-MWP generic probe assay in spite of a positive genotyping result. Concordance between the two laboratories was nearly 87%. Approximately 6% of samples that were positive or borderline when tested with the DIG-MWP generic probe assay were not detected with the HPV type-specific panel, perhaps representing very rare or novel HPV types. This new method is easier to perform than traditional generic probe techniques and uses more objective interpretation criteria, making it useful in studies of HPV natural history.Some types of human papillomavirus (HPV) are widely accepted as causative agents for cervical cancer (3,19). There are more than 40 HPV viral types that are commonly found in the genital tract, and approximately one-third of these are associated with cervical cancer and anal neoplasia. The anogenital HPV types are generally categorized as being either "high risk" or "low risk." High-risk types are associated with high-grade precancerous lesions and invasive cancer, while low-risk types are found in asymptomatic or benign conditions such as genital warts. However, the distribution and prevalence of types vary somewhat by geographic region and other demographic factors. Because the significance of the variation in type distribution is still being elucidated, studies of HPV epidemiology need to employ a methodology that can detect the entire spectrum of viral types. One of the most common means to detect and characterize new HPVs has been by PCR using consensus primers...
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