Detection of pathogenic leptospires by loop-mediated isothermal amplification targeting lipL32 Gene

Abstract

Background: Leptospirosis is a worldwide re-emerging infectious disease caused by pathogenic leptospires including Leptospira interrogans. Objective: In the present study, a loop-mediated isothermal amplification (LAMP) was developed to detect L. interrogans using lipL32 as a gene target. Material and Method: Four specific primers were designed based on the conserved region of lipL32 gene of various serovars of pathogenic leptospires. LAMP reaction was performed at 65°C for 1 hour. The LAMP products were detected by agarose gel electrophoresis and fluorescence dye. Results: The lipL32 LAMP assay showed highly specificity to the reference stains of L. interrogans serovar Autumnalis, Bataviae, Javanica, Pyrogenes, Icterohaemorrhagiae, and Saigon. No product was produced from non-pathogenic leptospire (L. biflexa), human, or Escherichia coli. The lower limit of detection analyzed by agarose gel electrophoresis and fluorescence dye visualization was 0.02 pg/μl which equivalent to 4 genomic equivalents/reaction. Moreover, the clinical strain of leptospires including pathogenic and intermediate group of L. interrogans were detected by lipL32 LAMP. Conclusion: The developed lipL32 LAMP is high specificity and sensitivity that can be applied to detect pathogenic leptospires in clinical samples. © 2015, Medical Association of Thailand. All rights reserved.