Journal of the Medical Association of Thailand = Chotmaihet thangphaet · 2015
Detection of Pathogenic Leptospires by Loop-Mediated Isothermal Amplification Targeting LipL32 Gene.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Seesom Weeranuch, Thongket Polphet, Rattanathanawan Kankanit, Mekseepralard Chantana, Sukhumsirichart Wasana
Аннотация
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.
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.