Rapid and simple identification of carbapenemase genes, bla NDM, bla OXA-48, bla VIM, bla IMP-14 and bla KPC groups, in Gram-negative bacilli by in-house loop-mediated isothermal amplification with hydroxynaphthol blue dye

Abstract

Carbapenem-resistant Enterobacteriaceae isolates by carbapenemase production are being reported globally with increasing frequency, leading to limited therapeutic options. We therefore developed a loop-mediated isothermal amplification method with hydroxynaphthol blue dye (LAMP-HNB) for rapid confirmation of blaNDM, blaOXA-48, blaVIM, blaIMP-14 and blaKPC groups. Sixty-two Enterobacteriaceae and Pseudomonas spp. isolates carrying various carbapenemase genes (28 blaNDM-1, 9 blaIMP-14a, 2 blaIMP-48, 1 blaIMP-1, 1 blaIMP-4, 1 blaIMP-9, 1 blaIMP-15, 4 blaVIM-2, 1 blaVIM-1, 1 blaIMP-14a & blaVIM-2, 7 blaKPC-2, 3 blaOXA-48 and 3 blaOXA-181) and 37 non-carbapenemase-producing Enterobacteriaceae isolates as confirmed by the PCR methods were included. Bacterial DNA was extracted by a simple boiling method. The LAMP-HNB method for each target gene was carried out using a set of six primers under isothermal condition at 65 °C in an ordinary water bath within 60 min and visual measurement of reaction by the change from violet to sky blue. This method had high efficiency (100% sensitivity and specificity) for identifying the blaNDM, blaOXA-48, blaVIM, blaIMP-14 and blaKPC groups compared with the PCR method. The HNB is easy to prepare, inexpensive and provides reliable results. Therefore, this method could be used as a confirmatory carbapenemase test in routine laboratory or for epidemiological purposes.