Genotypic detection of the bla<inf>CTX-M-1</inf> gene among extended-spectrum β-lactamase-producing Enterobacteriaceae

Abstract

© 2017 International Society for Chemotherapy of Infection and Cancer Objectives Extended-spectrum β-lactamases (ESBLs), a group of β-lactamase enzymes produced by bacteria in the family Enterobacteriaceae, are becoming a major problem in the healthcare community worldwide. Although many attempts have been made in the detection of ESBL-producing bacteria, the cost and speed of detection remains an important challenge. Therefore, this study aimed to develop a rapid, effective and affordable method for detection of the blaCTX-M-1 ESBL gene by a loop-mediated isothermal amplification (LAMP) technique. Methods Clinical ESBL-producing Enterobacteriaceae, including Escherichia coli and Klebsiella pneumoniae, were isolated and were used as representative strains. The double-disk synergy method was performed to detect ESBL-producing Enterobacteriaceae. Performance of the LAMP method in the detection of blaCTX-M-1 was compared with conventional PCR in terms of sensitivity and specificity. Results The developed LAMP method efficiently identified the presence of the blaCTX-M-1 gene in ESBL-producing Enterobacteriaceae. It provided similar results to conventional PCR, but the LAMP technique required only 20 min of testing time. The accuracy of the LAMP method was confirmed by restriction digestion, which showed the predicted size of the blaCTX-M-1 gene. In addition, the developed method was comparable with PCR that amplified only the target blaCTX-M-1 gene in terms of specificity, but LAMP was ca. 1000-fold more sensitive than PCR. Conclusions A rapid assay to detect ESBL-producing Enterobacteriaceae by a LAMP technique was developed in this study. The developed method is sensitive and suitable for rapid screening of blaCTX-M-1 in routine laboratories with limited resources.