DNA-EIA TO DETECT HIGH AND LOW-RISK HPV GENOTYPES IN CERVICAL LESIONSWITH E6 E7 PRIMER-MEDIATED MULTIPLEX PCR/

Background-Oncogenicity papillomavirus (HPV) DNA in premalignant and m alignant uterine cervical diseases is mainly induced by E6/E7 open rea ding frame (ORF). The presence of an oncogenic HPV DNA may be a diagno stic marker for the detection of cytologically negative smears. Aims-T o evaluate an original polymerase chain reaction enzyme immunoassay (P CR-EIA) for the detection and typing of oncogenic and non-oncogenic HP V types. Methods-The test was an original multiplex labelled PCR-EIA f or the detection and typing of oncogenic and nononcogenic HPV using th ree consensus sequence primers within the oncogenic E6/E7 ORF. One pri mer was dinitrophenyl (DNP) labelled and the DNP labelled amplimers co uld be further hybridised with specific biotinylated oligoprobes mixed in only two cocktails: oncogenic (16, 18, 31, 33, 35, 52, and 58) and nononcogenic (6 and 11) HPV types in only two wells; then biotinylate d oligoprobes were deposited in streptavidin-coated microplates. The P CR-EIA was validated on HPV plasmids (types 6, 11, 16, 18, 31, 33 35, 52, and 58) and used to evaluate cervical scrapes from 181 patients (m edian age 32 years) at high risk for cervical cancer. Results-HPV were detected in the cervical scrapes of 88 of 181 patients (48.6%); nine with non-oncogenic HPV (5.0%) and 79 with oncogenic HPV (43.6%) includ ing 29 coinfections with oncogenic and nononcogenic HPV. The number of oncogenic HPV infections increased with the presence of high grade le sions: 95.8% of the cervical scrapes from patients with high grade les ions contained oncogenic HPV compared with 32.1% of the specimens from patients without any lesions detectable by colposcopy and/or by cytol ogical examination of the cervical smears. Moreover, 60% of cervical s crapes exhibiting low grade lesions contained oncogenic HPV. Conclusio ns-This testis simple, specific, sensitive, safe, fast, reproducible, and easy to use in routine practice. Thus, it is possible to detect si multaneously on a simple cervical scrape, two kinds of HPV-oncogenic a nd non-oncogenic-in just two microplate wells with non-isotopic oligop robes.