Objective To develop a rapid method for detection Yersinia enterocolitica in patients with diarrhea. Methods Loop-mediated isothermal amplification （LAMP） reaction condition was optimized based on factorial experimental design. PCR and the developed LAMP methods were applied in clinical faecal samples and compared to evaluate its application. Results The optimal LAMP condition was containing 1.6 p~mol/L inner primer （FIP and BIP）, 1.6 mmol/L dNTPs, 2.0 mmol/L MgC12 in 25 p~l volumes, which was confirmed by factorial experiment analysis. And the amplification program was as follows： 63~C for 60 rain and then heated at 80~C for 10 min. Only genomic DNA from Yersinia enterocolitica strains was positively detected by LAMP, while no LAMP products were obtained when detecting non- Yersinia strains. LAMP method could detect Yersinia enterocolitica genomic DNA as low as 38.05 fg, while for the pure culture bacteria was 15 CFU/ml. In artificially infected faecal specimens, the detection limit was 150 CFU/g. With regard to the 539 clinical specimens from diarrhea patients, 13 were tested positive using the LAMP assay, and 11 were tested positive using the PCR assay, resulted in the sensitivity of 100% and specificity of 99.62%. Conclusions The optimized LAMP method was proved to be sensitive and specific, easy-to-use and low cost. LAMP is readily to applied in primary care clinics.
Journal Of Tropical Medicine