Table of Contents
International Journal of Bacteriology
Volume 2015, Article ID 147173, 6 pages
http://dx.doi.org/10.1155/2015/147173
Research Article

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

1Naval Medical Research Center, Silver Spring, MD 20910, USA
2Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA
3Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand

Received 13 October 2014; Revised 19 December 2014; Accepted 29 December 2014

Academic Editor: Christopher M. Parry

Copyright © 2015 Hua-Wei Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.