Group-specific differentiation between high- and low-risk human papillomavirus genotypes by general primer-mediated PCR and two cocktails of oligonucleotide probes.
In recent years, general primer-mediated PCR assays have been developed to detect a broad spectrum of human papillomavirus (HPV) genotypes. In this study, a procedure enabling a simple group-specific differentiation of high-risk (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -54, -56, and -58) and low risk (HPV-6, -11, -34, -40, -42, -43, and 44) HPVs following an HPV general primer-mediated (GP5+/GP6+) PCR is presented. By computer-assisted sequence analysis, oligonucleotides (30-mers) specific for 19 different HPV genotypes were selected from the internal part of the 150-bp GP5+/GP6(+)-amplified region. These oligo probes were tested for specificity in a Southern blot analysis of PCR products derived from the same panel of HPV types. No cross-hybridizations were found. The sensitivities of the oligo probes varied from the femtogram level for the well-amplified HPV types like HPV-16 and -18 to the picogram level for the less-well amplified HPV types like HPV-39 and -51. These sensitivities were reached when the oligo probes were applied both individually and in a cocktail. On the basis of these results, two cocktail oligo probes that enabled a specific and sensitive differentiation between low- and high-risk HPV types were composed.
Jacobs
M V
MV
Department of Pathology, Free University Hospital, Amsterdam, The Netherlands.
de Roda Husman
A M
AM
van den Brule
A J
AJ
Snijders
P J
PJ
Meijer
C J
CJ
Walboomers
J M
JM
D001483:Base Sequence; D002584:Cervix Uteri / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D015344:DNA Probes, HPV / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D005838:Genotype; D006801:Humans; D015336:Molecular Probe Techniques / Q000706:statistics & numerical data; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics* / Q000472:pathogenicity; D030361:Papillomavirus Infections / Q000209:etiology; D016133:Polymerase Chain Reaction / Q000706:statistics & numerical data; D012307:Risk Factors; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000209:etiology; D002583:Uterine Cervical Neoplasms / Q000209:etiology; D014773:Virology / Q000379:methods / Q000706:statistics & numerical data
D017931:DNA Primers; D015344:DNA Probes, HPV; D004279:DNA, Viral