Preparation Of PCR And Fluorescent Quantitative Polymerase Chain Reaction Kit For The Diagnosis Of Canine Leptospirosis

Leptospirosis is a zoonosis caused by the infection of pathogenic Leptospira.Canine leptospirosis is caused by Leptospira that usually infect Canine, asicterohemorrhagic, L.canicola, L.tarassovi etc. Dogs have a susceptibility to thisdisease, dogs infected with this disease has various clinical manifestations,diagnosis is difficult, so we need to diagnose this disease by laboratory testing.Common laboratory diagnosis methods are: the direct microscopic examination,isolation, serological test, animal inoculation, but these methods need a lot of timeor lack accuracy. Compared with these methods, polymerase chain reaction (PCR)method is more time-saving, has excellent specificity and sensitivity, and can beused for early diagnosis of leptospirosis.According to the conservative fragment of LigB gene which only appeared onpathogenic Leptospira, and compare with the common related sequences ofpathogenic Leptospira strains that usually infect canine, we designed pairs ofspecific, objective primers and probes, the fragment size is124bp. Filtrated theprobes and primers, optimized the reaction conditions and system, constructed theplasmid containing the target gene as a positive control, and verified it bysequencing of the plasmid. We established the leptospirosis PCR and fluorescencequantitative PCR method to diagnose canine, and then assembled into the kit whichcan realize fast and efficient diagnostic for canine leptospirosis. The sensitivity,specificity, repeatability and other parameters of the kit was tested, this experimentdetermined the storage conditions and period, collected and test clinical blood andurine samples and compared with the microscopic agglutination test, results wasgood.In recent years, the number of pet dogs is increasing, the number of workingdogs are also on the rise, canine have a susceptibility to canine leptospirosis, it could influence their health and the health of the host, the detection method can beapplied for early detection of canine leptospirosis, avoid further development anddiffusion of the disease.Cultivation of leptospira need weeks of time, the preparation process ofmedium is complex. MAT is gold standard method for diagnosis of leptospirosis,need to diagnose the living leptospira, so it is difficult to get the promotion andapplication. PCR method does not have to culture leptospira, and compared withserology methods, it can be used for early diagnosis of leptospirosis. In thisexperiment, the sensitivity of ordinary PCR can reach101, and the sensitivity offluorescent quantitative PCR is up to100, when detect leptospira bacteria liquid.