A Molecular-Beacon-Based Multiplex Real-Time PCR Assay To Distinguish Mycobacterium abscessus Subspecies and Determine Macrolide Susceptibility
Salvatore A. E. Marras, Liang Chen, Elena Shashkina, Rebecca M. Davidson, Michael Strong, Charles L. Daley, Barry N. Kreiswirth
Abstract
gene has dramatically impacted patient outcome. However, standard microbiology laboratories typically have limited diagnostic tools to distinguish M. abscessus subspecies, and the testing for macrolide resistance is often not done. Here, we describe the development of a real-time multiplex assay using molecular beacons to establish a robust, rapid, and highly accurate method to both distinguish M. abscessus subspecies and to determine which strains are susceptible to macrolides. We report a bioinformatic approach to identify robust subspecies sequence targets, the design and optimization of six molecular beacons to identify all genotypes, and the development and application of a 2-tube 3-color multiplex assay that can provide clinically significant treatment information in less than 3 h.