title: Simultaneous detection, typing and quantitation of oncogenic human papillomavirus by multiplex consensus real-time PCR.
abstract: A consensus multiplex real-time PCR test (PT13-RT) for the oncogenic human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 is described. The test targets the L1 gene. Analytical sensitivity is between 4 and 400 GU (genomic units) in the presence of 500 ng of human DNA, corresponding to 75,000 human cells. HPV types are grouped into multiplex groups of 3 or 4 resulting in the use of 4 wells per sample and permitting up to 24 samples per run (including controls) in a standard 96-well real-time PCR instrument. False negative results are avoided by (a) measuring sample DNA concentration to control that sufficient cellular material is present and (b) including HPV type 6 as a homologous internal control in order to detect PCR inhibition or competition from other (non-oncogenic) HPV types. Analysis time from refrigerator to report is 8 h, including 2.5 h hands-on time. Relative to the HC2 test, the sensitivity and specificity were respectively 98% and 83%, the lower specificity being attributable to the higher analytical sensitivity of PT13-RT. To assess type determination comparison was made with a reversed line-blot test. Type concordance was high (κ=0.79) with discrepancies occurring mostly in multiple-positive samples.
authors.affiliation: Unilabs Telelab AS, Skien, Norway. andrew.jenkins@hit.no
authors.forename: Andrew
authors.initials: A
authors.lastname: Jenkins
authors.forename: Anne-Gry
authors.initials: AG
authors.lastname: Allum
authors.forename: Linda
authors.initials: L
authors.lastname: Strand
authors.forename: Randi Kersten
authors.initials: RK
authors.lastname: Aakre
mesh_terms: D005260:Female; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D019562:Viral Load / Q000379:methods*