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

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 bla _NDM, bla _OXA-48, bla _VIM, bla _IMP-14 and bla _KPC groups. Sixty-two Enterobacteriaceae and Pseudomonas spp. isolates carrying various carbapenemase genes (28 bla _NDM-1, 9 bla _IMP-14a, 2 bla _IMP-48, 1 bla _IMP-1, 1 bla _IMP-4, 1 bla _IMP-9, 1 bla _IMP-15, 4 bla _VIM-2, 1 bla _VIM-1, 1 bla _IMP-14a & bla _VIM-2, 7 bla _KPC-2, 3 bla _OXA-48 and 3 bla _OXA-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 bla _NDM, bla _OXA-48, bla _VIM, bla _IMP-14 and bla _KPC 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.