Optimization and validation of LAMP technique for the detection of leptospiral DNA

Abstract

Leptospira spp. is the second most important abortifacient pathogen in livestock pro duction in Argentina, responsible for the loss of millions of dollars in the beef and dairy industry in the Pampas region (Cantón et al., 2022). It is crucial to have diagnostic tools that allow simple and effective detection of the agent, in order to implement neces sary measures to prevent spread of the pathogen and mitigate economic and public health consequences. The aim of this study was to develop a method for detecting leptospiral DNA based on loop-mediated isothermal amplification (LAMP) technique, designed for application in low-complexity laboratories: Lepto-LAMP. Molecular target of Lepto-LAMP was rrs gene (koizumi et al., 2012). After optimization, Lepto-LAMP in corporated calcein as an indicator and presented high analytical sensitivity (10 pg of DNA per reaction, or 0.1; 1 and 10 leptospires/mL in bovine serum, urine, and kidney macerate, respectively). Lepto-LAMP specifically detected DNA from pathogenic and intermediate strains of Leptospira spp. In a trial using hamsters (Mesocricetus auratus) as a model of animal leptospirosis, Lepto-LAMP allowed for the detection of a greater number of positive samples in infected animals compared with end-point PCR lipL32. Both molecular diagnostic techniques showed substantial agreement according to Cohen’s kappa coefficient. Employing clinical samples (serum, urine, and organs) from domestic and wild animals, Lepto-LAMP demonstrated high diagnostic sensitivity (100%, 95% CI:93.6–100%) and high diagnostic specificity (96.6%, 95% CI: 95.2–97.7%). Lepto-LAMP had a lower implementation cost compared to the end-point PCR, mak ing it more accessible for low-complexity laboratories. The use of Lepto-LAMP for the diagnosis of animal leptospirosis has great potential to complement the microscopic agglutination test (MAT) as it can be implemented in most non-specialized laborato ries and provides a short time to obtain results (between 75–90 minutes from DNA extraction to final result reading).