Rapid multiplex detection of the resistance genes mecA, vanA and vanB from Gram-positive cocci-positive blood cultures using a PCR-dipstick technique

Introduction. Among the causative agents of bloodstream infections (BSIs), methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococcus (VRE) are the key causative pathogens. Their rapid detection directly from Gram-positive cocci-positive blood culture specimens will promote timely treatment and help to implement effective infection control measures.Aim. We aim to develop a PCR-dipstick technique for the rapid detection of MRSA and VRE directly from positive blood culture specimens.Methodology. PCR-dipstick is a PCR-based multiplex detection technique where DNA-DNA hybridization is employed, and the results are interpreted with the naked eye. It was designed to target three drug resistance genes: mecA in MRSA and vanA/vanB in VRE from positive blood culture specimens. A total of 120 clinical isolates were used to evaluate the sensitivity and specificity of PCR-dipstick. Then, PCR-dipstick was examined for MRSA and VRE detection directly from positive blood cultures.Results. PCR-dipstick showed 100 % sensitivity and specificity in detecting mecA, vanA and vanB genes directly from bacterial colonies in comparison with multiplex PCR for genomic DNA followed by agarose gel electrophoresis. Further, it could differentially detect multiple resistant genes in pooled bacterial colonies (n=10). Ultimately, PCR-dipstick could detect MRSA and VRE in positive blood cultures in ~3 h.Conclusion. The results of the current study substantiate that PCR-dipstick can be used as an efficient detection system for MRSA and VRE directly from Gram-positive cocci-positive blood cultures. Its affordability and rapidity indicate that PCR-dipstick can be an effective tool for controlling nosocomial pathogens.