Multiplex real-time PCR assay for the detection of extended-spectrum beta-lactamase and carbapenemase genes using melting curve analysis

作者：Singh Prashant; Pfeifer Yvonne; Mustapha Azlin^*

来源：Journal of Microbiological Methods, 2016, 124: 72-78.

DOI：10.1016/j.mimet.2016.03.014

• Antibiotic resistance
• ESBL
• Enterobacteriaceae

Real-time PCR melt curve assays for the detection of beta-lactamase, extended-spectrum p-lactamase and carbapenemase genes in Gram-negative bacteria were developed. Two multiplex real-time PCR melt curve assays were developed for the detection of ten common beta-lactamase genes: NaKPC-like. NaOXA-48-like, bla(NDM-like), bla(VIM-like), bla(IMP-like), bla(CTX-M-1+2-group), bla(CMY-like), blaACC-liker bla(SHV-like) and bla(TEM-like). The assays were evaluated using 25 bacterial strains and 31 DNA samples (total n = 56) comprising different Enterobacteriaceae genera and Pseudomonas spp. These strains were previously characterized at five research institutes. Each resistance gene targeted in this study generated a non-overlapping and distinct melt curve peak. The assay worked effectively and detected the presence of additional resistance genes in 23 samples. The assays developed in this study offer a simple, low cost method for the detection of prevalent beta-lactamase, ESBL and carbapenemase genes among Gram-negative pathogens.

• 出版日期2016-5