Development of a simplified assay for detection of van gene harbored enterococci using the automated BD MAX platform

Abstract We developed and evaluated of multiplex real-time PCR assay for detection of vancomycin-resistant genes (vanA, vanB, vanC1 and vanC2/C3) using the new, fully automated BD MAX platform. Ct value analyses of real-time PCR simultaneous repeatability test have showed the usefulness; coefficient of variation: CV (%) were determined 2.09%, 1.72%, 1.41% and 1.52% with vanA, vanB, vanC1 and vanC2/C3, respectively. We also evaluated with 43 strains of enterococci were characterized by conventional PCR method; 4/4 for vanA-positive, 14/14 for vanB-positive, 1/1 for vanB plus vanC1-positive, 6/6 for vanC1-positive, 4/4 for vanC2/C3- positive and 14/14 for all-van gene-negative strains were identified correctly. This assay was automatically performing before and after PCR operations previously done manually by operator, such as DNA extraction, sample dispensing and gel electrophoresis or the ethidium bromide dyeing. As a result, work burden and the risk of the contamination were largely reduced and were shortened to about half for measurement time. We conclude that this assay could greatly contribute to efficient and rapid detection of vancomycin-resistant genes.