Development and Validation of a Clinical Laboratory Improvement Amendments-Compliant Multiplex Real-Time PCR Assay for Detection of Genes.

Increased use of colistin in both human and veterinary medicine has led to the emergence of plasmid-mediated colistin resistance ( genes). In this study, we report the development of a real-time PCR assay using TaqMan probe-based chemistry for detection of genes from bacterial isolates. Positive control isolates harboring and yielded exponential amplification curves with the assay, and the amplification efficiency was 98% and 96% for and , respectively. Each target gene could be reproducibly detected from a sample containing 10 cfu/mL of -harboring bacteria, and there was no cross-reactivity with DNA extracted from several multidrug-resistant bacteria harboring other resistance genes, but lacking genes. Both sensitivity and specificity of the real-time PCR assay were 100% in a method validation performed with a set of 25 previously well-characterized bacterial isolates containing -positive and -negative bacteria. This newly developed assay is a rapid and sensitive tool for detecting emerging genes in cultured bacterial isolates. The assay was successfully validated according to quality standards of the Clinical Laboratory Improvement Amendments (CLIA).