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.