BACKGROUND: Nucleic 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. OBJECTIVES: This study compares five different automated extraction platforms for the extraction of norovirus RNA from stool and cytomegalovirus (CMV) DNA from plasma samples. STUDY DESIGN: Norovirus 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. RESULTS: Of 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. CONCLUSIONS: The five automated extraction platforms yielded comparable results. However, the extraction performance was found to be impaired by inhibitory substances in stool samples.
BACKGROUND: Nucleic 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. OBJECTIVES: This study compares five different automated extraction platforms for the extraction of norovirus RNA from stool and cytomegalovirus (CMV) DNA from plasma samples. STUDY DESIGN: Norovirus 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. RESULTS: Of 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. CONCLUSIONS: The five automated extraction platforms yielded comparable results. However, the extraction performance was found to be impaired by inhibitory substances in stool samples.
Authors: Deirdre L Church; Lorenzo Cerutti; Antoine Gürtler; Thomas Griener; Adrian Zelazny; Stefan Emler Journal: Clin Microbiol Rev Date: 2020-09-09 Impact factor: 26.132
Authors: Ambroos Stals; Elisabeth Mathijs; Leen Baert; Nadine Botteldoorn; Sarah Denayer; Axel Mauroy; Alexandra Scipioni; Georges Daube; Katelijne Dierick; Lieve Herman; Els Van Coillie; Etienne Thiry; Mieke Uyttendaele Journal: Food Environ Virol Date: 2012-11-04 Impact factor: 2.778
Authors: Jens Verheyen; Kseniya Maizus; Eugen Feist; Zebulon Tolman; Elena Knops; Jasemine Saech; Lydia Spengler; Tim Waterboer; Gerd R Burmester; Michael Pawlita; Herbert Pfister; Andrea Rubbert-Roth Journal: Med Microbiol Immunol Date: 2015-02-13 Impact factor: 3.402