Multiplex real-time PCR assays to detect Stenotrophomonas maltophilia carrying sul1, sul2, and sul3 genes.

Nosocomial infections caused by Stenotrophomonas maltophilia resistant to SXT are increasingly reported worldwide. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of the ssrA and sul1, sul2 and sul3 genes was first established. The assays were performed on a Roche LightCycler® 480 II system. The results for target and non-target amplification showed that the multiplex real-time PCR assays were specific, the limit of detection for each target was 10 copies per 20 μL reaction volume, the assays were linear over six log dilutions of the target genes (r > 0.99), and the Ct values of the coefficients of variation for intra- and interassay reproducibility were <5%. The sensitivity for the target DNA in simulated blood samples was 10 CFU/mL. The multiplex real-time PCR assays showed 100% concordance with conventional PCR when tested against 20 SXT-susceptible and 20 SXT-resistant S. maltophilia from clinical samples. Therefore, the multiplex real-time PCR is a rapid, affordable and sensitive assay for direct detection of the ssrA and sul1, sul2 and sul3 genes.