The next-generation Hybrid Capture® High-Risk HPV DNA assay on a fully automated platform

Background: A next-generation diagnostic system has been developed at QIAGEN. The QIAensemble(TM) system consists of an analytical subsystem (JE2000(TM)) that utilizes a re-engineered Hybrid Capture(R) chemistry (NextGen) to maintain the high level of clinical. sensitivity established by the digene High-Risk HPV DNA Test(R) (HC2), white creating improved analytical specificity as shown both in plasmid-based analyses and in processing of clinical specimens. Study Design: Limit-of-detection and cross-reactivity experiments were performed using plasmid DNA constructs containing multiple high-risk (HR) and tow-risk (LR) HPV types. Cervical specimens collected into a novel specimen collection medium, DCM, were used to measure stability of specimens, as well as analytical specificity. Signal carryover, instrument precision, and specimen reproducibility were measured on the prototype JE2000 system using the automated NextGen assay. Results: The Limit of Detection (LOD) is <1000 copies of HPV 16 plasmid in the automated assay. No cross-reactivity (signal above cutoff) was detected on the automated system from any of 13 LR types tested at 107 copies per assay. Within-plate, plate-to-plate, and day-to-day performance in the prototype system yielded a CV of 20%. No indication of target carryover was found when samples containing up to 10(9) copies/ml of HPV DNA type 16 were processed on the JE2000 instrument. In an agreement study with HC2, 1038 donor cervical specimens were tested in both the manual NextGen assay and HC2 to evaluate agreement between the two tests. After eliminating discrepant specimens that were adjudicated by HR-HPV genotyping, the adjudicated positive agreement was 98.5% (95% CI: 94.6, 99.6). Conclusions: The JE2000 prototype system automates NextGen assay processing, yielding accurate, reproducible, and highly specific results with both plasmid analytical model tests and cervical specimens collected in DCM. The final system will process more than 2000 specimens in an 8-hour shift, with fully continuous loading. (C) 2009 Elsevier B.V. All rights reserved.