Detection of Pathogenic Leptospires by Loop-Mediated Isothermal Amplification Targeting LipL32 Gene.

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 ofpathogenic leptospires. LAMP reaction was performed at 65 °C for 1 hour The LAMP products were detected by agarose gel electrophoresis andfluorescence 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 andfluorescence 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.