Development of a Multiplex Polymerase Chain Reaction-Based DNA Lateral Flow Assay as a Point-of-Care Diagnostic for Fast and Simultaneous Detection of MRSA and Vancomycin Resistance in Bacteremia.

To reduce high mortality and morbidity rates, timely and proper treatment of methicillin-resistant (MRSA) bloodstream infection is required. A multiplex polymerase reaction (mPCR)-based DNA lateral flow assay (MBDLFA) was developed as a point-of-care diagnostic for simultaneous identification of , methicillin resistance, and vancomycin resistance directly from blood or blood cultures. A mPCR was developed to detect , A, and A/B; its sensitivity, specificity, and limit of detection (LOD) were determined. The developed reaction was further modified for use in MBDLFA and its sensitivity for detection of target genes from artificially inoculated blood samples was checked. The optimized mPCR successfully detected , A, and A/B from genomic DNA of bacterial colonies with LODs of 10, 10, and 10 CFU/mL, respectively. The reaction was sensitive and specific. The optimized mPCR was used in MBDLFA that detected , A, and A/B with LODs of 10, 10, and 10 CFU/mL, respectively, directly from artificially inoculated blood. The developed MBDLFA can be used as a rapid, cheap point-of-care diagnostic for detecting , MRSA, and vancomycin resistance directly from blood and blood cultures in ~2 h with the naked eye. This will reduce morbidity, mortality, and treatment cost in bacteremia.