Detection of Pathogenic Leptospires by Loop-Mediated Isothermal Amplification Targeting LipL32 Gene
Weeranuch Seesom MSc*, Polphet Thongket MSc*, Kankanit Rattanathanawan PhD**, Chantana Mekseepralard PhD***, Wasana Sukhumsirichart PhD*
Affiliation : * Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand ** Innovative Learning Center, Srinakharinwirot University, Bangkok, Thailand *** Department of Microbiology, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand
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.
Keywords : Loop-mediated isothermal amplification, LAMP, Leptospira interrogans, LipL32, Detection, Leptospirosis
