Development of real-time loop-mediated isothermal amplification (RealAmp) method for sensitive and rapid detection of pathogenic and nonpathogenicLeptospira

Abstract

Leptospirosis is a zoonotic disease that is spread through water contaminated with the spirochete Leptospira. In our study, RealAmp (real‐time loop‐mediated isothermal amplification) was developed to distinguish between pathogenic and nonpathogenic Leptospira spp. Melt curve analysis of RealAmp showed two distinct melt curves for pathogenic and nonpathogenic Leptospira spp with a Tm value of 90 ± 2 and 88 ± 2°C. The sensitivity of the developed method is as low as 1 pg μl−1 of Leptospira DNA. Specificity was achieved to distinguish Leptospira species using the marker genes (lipL32 and lipL21). Different environmental water samples collected from cattle shelter, pisciculture and stagnant water were tested using this RealAmp method. Pisciculture water samples were found to be highly contaminated with both pathogenic and nonpathogenic Leptospira spp (50%), followed by cattle shelter and stagnant water samples (15%). Significance and Impact of the Study The occurrence of pathogenic Leptospira in the environment is a threat to humans and animals. Molecular‐based differentiation of the pathogenic and nonpathogenic Leptospria is essential for environmental monitoring; however, the currently available detection methods fail to distinguish between these species. We report here a real‐time loop‐mediated isothermal amplification‐based method that differentiates between the pathogenic and nonpathogenic Leptospira based on the melt curve analysis of the marker genes lipL32 and lipL21. The method was successfully tested with a variety of environmental water samples to study the prevalence of Leptospira in the environment.

An overview on the molecular diagnosis of animal leptospirosis

“…This simplicity and low cost enhance its applicability for on‐field screening of pathogens and for limited resourced laboratories (Monica et al . 2019). Regarding its use as a diagnostic tool for animal leptospirosis, it has been successfully applied for detecting leptospiral DNA on cattle and buffalo (Suwancharoen et al .…”

Sensitive and rapid detection of Ortleppascaris sinensis (Nematoda: Ascaridoidea) by loop-mediated isothermal amplification

“…In this study, a real-time turbidimeter and colorimetric indicator were used to inspect the LAMP products. The positive curve monitored under a Loopamp real-time turbidimer can be seen directly through the screen during the real-time LAMP (Monica, Rathinasabapathi & Ramya, 2019; Liu et al, 2012). Furthermore, the presence of loop primers can increase the efficiency of amplification and make the detection more rapidly.…”

Molecular and serological diagnostic of leptospirosis: a review (2014–2020)

“…The biological samples used in most of these studies were blood or its fractions (serum or plasma) (Ahmed et al, 2014;Denipitiya et al, 2016;Gokmen et al, 2016;Nhan et al, 2014;Waggoner et al, 2015) and urine (Esteves et al, 2018;Podgorsek et al, 2020;Suwancharoen et al, 2016). Rathinasabapathi, & Ramya, 2019;Nhan et al, 2014;Suwancharoen et al, 2016;Waggoner et al, 2015).…”

“…As an alternative to the high cost of implementing real-time PCR in the routine, the RPA technique (Ahmed et al, 2014) proved to be accurate, with sensitivity and specificity of 94.7% and 97.7%, respectively. The 16SrRNA-PCR-RFLP (Gokmen et al, 2016) and RealAmp (Monica et al, 2019) techniques showed promising results, with an early and accurate diagnosis, which distinguished pathogenic and nonpathogenic species from Leptospira spp. Moreover, a novel multiplex PCR assay has been developed for identifying Orientia tsutsugamushi, Rickettsia typhi and Leptospira spp., causative agents of scrub typhus, murine typhus, and leptospirosis (Sea-Liang et al, 2019).…”