Highly Sensitive Loop-Mediated Isothermal Amplification for the Detection of Leptospira

Abstract

Leptospirosis is a worldwide zoonosis caused by an infection with the pathogenic species of Leptospira. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the DNA of Leptospira spp. Six sets of primers targeting the gene of the subsurface protein, lipL32, were evaluated for their detection sensitivity. The best primer set detected less than 25 copies of lipL32 per reaction of both plasmid DNA template and purified leptospiral genomic DNA. By combining primers targeting lipL32 with the previously published primer set targeting lipL41, the sensitivity of the assay was improved to 12 copies of L. interrogans. The specificity of the LAMP assay was evaluated by using the genomic DNA from other clinically encountered bacterial species such as different strains of Orientia tsutsugamushi, Rickettsia typhi, Rickettsia conorii, Rickettsia rickettsii, Coxiella burnetii, and Bartonella bacilliformis. These genomic DNA samples were all negative in our LAMP assay. The sensitivity of the LAMP assay was very similar to that of quantitative real time PCR. Several detection methods for the amplified product of LAMP assay were performed to demonstrate the simplicity of the assay. In summary, our results have suggested that this assay is rapid, robust, and easy to perform and has the potential to be used in endemic locations.

Figure 1

Different methods to detect LAMP products. Reaction products were visualized on GelRed stained gel (a) or visualized directly by including hydroxy naphthol blue (b) or SYBR green (c) in the reaction. A mixture of primer sets L32-5 and L41 was used in the reaction with different copies of genomic DNA. Reactions were carried out at 63°C for 60 min. Marker, 100 bp ladder, lanes 1 to 5, reaction mixture containing 100, 50, 25, and 12, and no copies of genomic DNA.