Cloning of the lipL41 Gene for Preparation of Positive Control to Detection of Pathogenic Leptospires
Leptospirosis is one of the most important zoonosis with worldwide distribution. lipL41 is an immunogenic outer membrane protein found in pathogenic Leptospira species and may be used in diagnostic method and also can be a good candidate for recombinant vaccine against leptospirosis. The aim of this study was designing a positive control for molecular diagnosis of pathogenic Leptospira spp. by PCR based on lipL41 gene. Five pathogenic serovars and one saprophytic species were used in this study. The serovars were subcultured into the selective culture medium (EMJH) and then the genomic DNA extracted. The lipL41 gene amplified using the specific primers. The PCR product were ligated in pTZ57R/T vector and transformed in competent E. coli Top10 cells. The confirmation of the recombinants was made by picking the white colonies and carrying out colony PCR amplification of the gene. The recombinant plasmids were extracted using a commercial extraction kit. PCR amplification of the lipL41 gene using the specific primers resulted in a 1065 bp in all five pathogenic serovars tested. No PCR products were amplified from the non-pathogenic L. biflexa. Positive colonies plasmid vector was isolated from cells by kit. PCR test was carried out with positive control and PCR products were observed on gel electrophoresis. Due to slow growth of Leptospira، and because of limited diagnostic capacity using a rapid and accurate molecular tests like PCR with a positive control to confirm its accuracy is essential. Therefore، the cloned lipL41 gene in this study that has been prepared in Leptospira reference laboratory can be used as a positive control in all laboratories using the PCR test.
Journal of Veterinary Microbiology, Volume:10 Issue:2, 2015
123 - 129
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