Extraction of viral nucleic acids: Comparison of five automated nucleic acid extraction platforms

Abstract

BackgroundNucleic acid extraction has a major impact on the reliability of results in routine molecular diagnostics. Optimal isolation of nucleic acids and removal of inhibitors are essential. ObjectivesThis study compares five different automated extraction platforms for the extraction of norovirus RNA from stool and cytomegalovirus (CMV) DNA from plasma samples. Study designNorovirus positive stool samples and CMV positive plasma samples were aliquoted and analyzed using five different automated platforms (easyMAG, bioMerieux; m2000sp, Abbott; MagNA Pure LC 2.0, Roche; QiaSymphony, Qiagen; sample preparation module of the VERSANT kPCR Molecular System, Siemens). Similar sample input and output volumes, the identical real-time PCR cycler, and the identical assays for amplification and detection for norovirus RNA and CMV DNA, respectively, were chosen. ResultsOf 39 stool samples, 36 tested positive for norovirus RNA with all extraction platforms. The three discrepant samples showed inhibition after extraction with at least one platform. Only with the VERSANT platform all samples tested positive for both the target RNA and the internal controls. Of 42 plasma samples, 27 gave quantifiable results for CMV DNA with all extraction platforms. There was significant variance between viral concentrations when different extraction platforms were compared. The majority of the 15 discrepant samples showed low viral concentrations. The internal control of the CMV assay gave positive results for all samples tested below the limit of quantification. ConclusionsThe five automated extraction platforms yielded comparable results. However, the extraction performance was found to be impaired by inhibitory substances in stool samples.