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.

Molecular typing on Leptospira interrogans isolates from Jiangxi province,by pulsed-field gel electrophoresis / 中华流行病学杂志

Objective To perform a molecular epideminlogical investigation on the types of Leptospira interrogans isolates from leptospirosis patients and animal hosts in Jiangxi province,using a pulsed-field gel electrophoresis (PFGE).Methods The extracted chromosomal DNA from leptospiral isolates were digested with restriction endonuclease Not Ⅰ and the DNA segments were separated by using PFGE.By BiOnurerics V4.0 software and 75% similarity as the standard,the obtained PFGE images from leptospiral isolates were managed to establish a digitization database and then the PFGE maps of leptospiral isolates were compared with those of reference standard strains belonging to 15 serovars in 15 serogroups of L.interrogans,for cluster analysis.Results 139 strains of L.interrogans isolated from different areas of Jiangxi province were classified into 46 PFGE types.Among the PFGE types,LepNot Ⅰ.0071,LepNotⅠ.0072 and LepNot Ⅰ .0043 were the predominant types that accounting for 28.06%,15.11% and 7.19% of all the leptospiral isolates,respectively.The PFGE maps from 84.89% (118/139) of the 139 leptospiral isolates were found to basically match those of 6 reference standard strains belonging to 6 serovar in 6 serogroups of L.interrogans.In the 118 matched ieptospiral isolates,32.37% (45 strains),15.83% (22 strains) and 15.11% (21 strains)belonged to sero-groups Icterohaemorrhagiae serovar Lai,sero-groups Australis serovar Australis and sero-group Javanica serovar Javanica,respectively.Conclusion PFGE seemed a fast,accurate and effective method for typing of L.interrogans isolates.Serogroup Icterohaemorrhagiae serovar Lai and followed by serogroup Australis serovar Australis as well as serogroup Javanica serovar Javanica were the predominant L.interrogans species in humans and animal hosts in Jiangxi province.

Establishment of standardized method for pulsed-field gel electrophoresis on Leptospira interrogans and analysis on their patterns / 中华流行病学杂志

<p><b>OBJECTIVE</b>To establish a standardized operation procedure for pulsed-field gel electrophoresis (PFGE) on Leptospira interrogans as well as a figure digital database to develop the Chinese representative reference strains.</p><p><b>METHODS</b>Under the characteristics of strains and referring to the other SOPs of PFGE on pathogens provided by CDC and PulseNet Asia Pacific, genomic chromosome DNA purification, restriction endonuclease digestion and the parameters for running PFGE were optimized.</p><p><b>RESULTS</b>Not I digestion patterns of leptospiral genome for the Chinese representative strains were established and partial isolates of serogroup icterohaemorrhagiae from the leptospirosis surveillance in Sichuan and Anhui provinces were analyzed by PFGE. Results showed that each of all the 15 Chinese representative strains had a unique pattern. 91.67% (22/24) of the 24 isolates identified as serogroup icterohaemorrhagiae matched to the map of the reference strain 56601 (serogroup icterohaemorrhagiae serovar lai).</p><p><b>CONCLUSION</b>The PFGE figures were clear with high resolution and the fragments were equally distributed by this standardized operating procedure so as to reveal the molecular-genetic characteristics of Leptospira interrogans. The patterns had high relativity with the serological identification and seemed to be very important for genetic analysis of strains in studying the outbreak of leptospirosis.</p>