Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay

Abstract

A gene amplification method that combines PCR with an enzyme immunoassay (PCR-EIA) for quantitation of amplified DNA was developed for the detection of human papillomavirus (HPV). Samples were amplified with consensus primers MY09 and MY11. Amplified DNA products were reacted in solution with type-specific nested RNA probes labelled with digoxigenin-11-UTP. Hybrids were captured on a microtiter plate coated with an antidigoxigenin antibody. Bound DNA-RNA hybrids were quantitated by the addition of an alkaline phosphatase-labelled monoclonal antibody directed against DNA-RNA hybrids and a fluorogenic substrate. The detection limit of PCR-EIA was six copies of HPV type 18 DNA in the original specimen. The assay was used to assess HPV infection of the uterine cervixes of 65 women referred to a colposcopy clinic. In 66 cervicovaginal lavage specimens, all 23 HPV strains detected by a standard isotopic PCR assay were also detected by the PCR-EIA (sensitivity, 100%; 95% confidence interval, 85.2 to 100%). Forty-two of the 43 samples that did not contain HPV types 6/11, 16, 18, 31, 33, 35, and 45 were also negative by PCR-EIA, for a specificity of 97.7%. Low-level cross-reactivity was encountered between HPV types 18 and 45 as well as between types 33 and 58. PCR-EIA provides a convenient means of objectively measuring PCR-amplified HPV DNA from common genital HPV types.

Prevalence and viral load of oncogenic human papillomavirus (HPV) in pterygia in multi‐ethnic patients in the Malay Peninsula

“…Each of the extracted DNA samples was amplified with PC03 and PC04 human β ‐globin primers (Coutlée et al. ) to ensure the DNA integrity and to rule out false‐negative results for the subsequent HPV DNA PCR detection. The following were mixed in each 0.2 ml microfuge tube: 1× PCR buffer, 2.5 m m MgCl 2 , 0.2 m m dNTPs, 0.5 μ l each of 10 pmol/ μ l forward and reverse primers and 1.0 μ l of DNA template.…”

“…The primers used for the first round of nested PCR were MY09 and MY11, while the primers for the second round were GP5+ and GP6+ (Coutlée et al. ). The volumes and concentrations of reagents used were identical to those used for the β ‐globin PCR; except that for the second round of PCR, 2 μ l of undiluted first‐round PCR products were used as the template.…”

A general primer GP5+/GP6(+)-mediated PCR-enzyme immunoassay method for rapid detection of 14 high-risk and 6 low-risk human papillomavirus genotypes in cervical scrapings

“…In this study we have used three times the mean OD value of the PCR negative controls as a cutoff point. Although there is no international standardization for determining this value for HPV PCR-EIA (1,3,17,20), this cutoff value revealed a 100% agreement between EIA results and results from the radioactive method. However, to get more insight in determining a cutoff point, we are in the process to analyze a series of HPVnegative scrapings with the PCR-EIA.…”

“…Recent data indicate that the application of an enzyme im-munoassay (EIA) to detect PCR products can be an appropriate nonradioactive alternative (1,3,14,17,20). Therefore, we aimed in this study to develop an HR/LR HPV PCR-EIA for the detection of 14 HR and 6 LR HPV genotypes as mentioned above.…”

Nucleic acid tests for the diagnosis of sexually transmitted diseases

“…The test was accurate and reliable in an interlaboratory agreement study [42]. More recently, HPV PCRs have been described that are more sensitive than the original hybrid capture [43,44]. The presence of more than 70 viral types, including at least 20 found in the genital mucosa, led investigators to design consensus primers that amplify highly conserved viral gene fragments in a single reaction.…”