Validation of a nested PCR assay UMELOSA((R))HCV CUALITATIVO for the detection of Hepatitis C virus.

摘要

An analysis of the performance characteristics of the UMELOSA((R))HCV CUALITATIVO assay for the detection of Hepatitis C virus (HCV) RNA in human serum or plasma is presented. This qualitative in vitro diagnostic test is performed in three steps: (a) extraction of viral RNA, (b) reverse transcription of target RNA to generate complementary DNA followed by a polymerase chain reaction assay coupled with a second round of amplification, and finally (c) fluorescent detection of the amplified DNA by hybridization in a solid phase of an ultramicroplate coated with a complementary to amplified DNA probe.CONSIDERING THE ASSAY AS A LIMIT TEST FOR THE CONTROL OF IMPURITIES, THE FOLLOWING ANALYTICAL PERFORMANCE PARAMETERS WERE EVALUATED: specificity, detection limit and robustness. A comparative evaluation of the clinical performance and detection limit of our kit and the commercial AMPLICOR((R))HCV test, version 2.0, was also included in the validation protocol.The assay had a good specificity and did not cross-react with the non-HCV analyzed positive samples. The 95% detection limit was of 101.7IU/ml with 95% confidence interval from 81.0 to 162.8IU/ml. The UMELOSA((R))HCV CUALITATIVO meets the minimal sensitivity requirements for a single unit blood testing of 5000 and 1250IU/ml, defined by the Paul-Ehrlich-Institute in Germany and the Food and Drug Administration in USA, respectively.Compared with the commercial AMPLICOR, the test gave identical results for all analyzed positive and negative samples. In robustness studies there was no cross-contamination between negative samples and these same samples spiked with 10000IU/ml of HCV-RNA.