NDLI: Nonisotopic Detection of Human Papillomavirus DNA in Clinical Specimens Using a Consensus PCR and a Generic Probe Mix in an Enzyme-Linked Immunosorbent Assay Format

Content Provider	PubMed Central
Author	Kornegay, J. R. Shepard, A. P. Hankins, C. Franco, E. Lapointe, N. Richardson, H. Coutleé, F.
Copyright Year	2001
Abstract	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 β-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.
Related Links	http://dx.doi.org/10.1128/jcm.39.10.3530-3536.2001
Ending Page	3536
Page Count	7
Starting Page	3530
File Format	PDF
ISSN	00951137
e-ISSN	1098660X
Journal	Journal of Clinical Microbiology
Issue Number	10
Volume Number	39
Language	English
Publisher	American Society for Microbiology
Publisher Date	2001-10-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Microbiology (medical) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Microbiology (medical)

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