Molecular typing of Leptospira interrogans strains isolated from Rattus tanezumi in Guizhou Province, Southwest of China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To identify and type three leptospires isolated from Rattus tanezumi in Guizhou Province by using three molecular techniques (PFGE, MLVA, and MLST), reveal the molecular characteristic of causative agents of local leptospirosis and evaluate these three molecular methods based on their detection resolution and efficiency.</p><p><b>METHODS</b>Three Leptospira strains were isolated from the kidney of Rattus tanezumi and cultured with EMJH medium. PFGE, MLVA, and MLST assays were applied to type the three strains isolated from Rattus tanezumi in Guizhou Province.</p><p><b>RESULTS</b>PFGE, MLVA, and MLST typing showed that the three leptospiral isolates matched with leptospiral serogroup Icterohaemorrhagiae serovar Lai. The findings of the genotyping methods were consistent. MLVA and MLST defined genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes, and the findings of molecular typing were also consistent with those of traditional techniques.</p><p><b>CONCLUSION</b>Three leptospiral isolates from Guizhou Province matched with leptospiral serogroup Icterohaemorrhagiae serovar Lai, and PFGE, MLVA, and MLST, as reliable molecular techniques for identifying and typing of Leptospira interrogans, would contribute to the active surveillance, outbreak investigation and source tracking for leptospirosis in Guizhou Province.</p>

Establishment and application of TaqMan Real-time PCR for the detection of pathogenic Leptospira species / 中华流行病学杂志

Objective To develop and evaluate a TaqMan Real-time PCR method for the detection of pathogenic Leptospira species.Methods rrs gene of part fragment on 16S rRNA was used to design primers and TaqMan probe.The target gene was cloned into vector pMD19-T in order to make the standard curve and be used for quality control.To determine the specificity and specificity,DNA from Chinese Leptospira strains belonging to 15 pathogenic reference strains,21non-pathogenic reference strains,and 50 different serotypes of pathogenic isolates as well as 27 other micro-organisms were included in this study.Eight serial DNA dilutions from pathogenic Leptospira and DNA from 25 kidney tissues were detected by Real-time PCR and conventional PCR simultaneously.Results A Real-time PCR methodology was developed and optimised.All the pathogenic Leptospira gave a positive amplification.Non-pathogenic Leptospira and all the other micro-organisms were not amplified.The plasmid sensitivity of Real-time PCR and conventional PCR were 10 copy/μl and 104 copy/μl respectively.The DNA sensitivity of Real-time PCR and conventional PCR were 100 fg/μl and 1 ng/μl respectively.The kidney tissue detection of the two methods appeared to be exactly the same.Conclusion This research project successfully developed a Real-time PCR methodology with better sensitivity and specificity for the identification of pathogenic Leptospira,using the rrs gene.