Development of duplex loop-mediated isothermal amplification (dLAMP) for detection of carbapenem antibiotic-resistant genes KPC and NDM

Abstract

Carbapenems are regarded as a last-resort option for treating a wide range of Gram-positive and Gram-negative bacterial infections. Unfortunately, the prevalence of carbapenem-resistant genes has been on the rise among patients. Some factors contribute to this trend, including prolonged hospital stays, prior antibiotic usage, inappropriate or insufficient antibiotic treatment, and contamination through wounds or feces. The emergence of carbapenem-resistant (CR) genes gained significant attention, particularly after 2005. These CR genes are frequently carried on mobile genetic elements such as plasmids or transposons, enabling their transmission to other bacteria. Klebsiella pneumoniae carbapenemase (KPC) and New delhi metallo-β-lactamase (NDM) stand out as the most prevalent CR genes, both in Southeast Asian countries and worldwide. These genes exhibit numerous subtypes, with 123 and 43 subtypes for KPC and NDM, respectively. Hence, in this study, our focus was on KPC and NDM as the most widespread CR genes that have been responsible for numerous global outbreaks in recent times. We developed universal primers for KPC and NDM genes, aiming to detect the subtypes using loop-mediated isothermal amplification (LAMP) techniques. Additionally, we developed a duplex-LAMP assay which is capable of simultaneously detecting both genes in a single reaction. As a result, the developed dLAMP can detect KPC and NDM in a single reaction using an optimum temperature and incubation time of 55 minutes at 65°C temperature. For visualization, using hydroxyl naphthol blue (HNB) which changed from violet (negative) to blue (positive). With optimum MgSO4 and HNB concentration, 6.5 mM, and 180 mM, showed the highest absorbance at 650 nm. The developed universal primers KPC and NDM proved to be specific only for detecting both genes using PCR. Furthermore, the sensitivity of developed dLAMP in the detection of both genes was ten times higher compared to traditional PCR with approximately 1 hour to determine the positive results of dLAMP.