Development of a Taqman multiplex real-time PCR to detect Leptospira spp.through bioinformatics analysis of the outer membrane protein

Leptospirosis is a zoonotic disease synonymously occurring in tropical climate countries. The etiology of the disease is due to bacteria from genus Leptospira. Higher number of cases reported worldwide indicate the disease is not easily eradicated. Leptospirosis shares the most common febrile symptoms such as dengue, Zika and yellow fever thus making it difficult to differentiate the disease at an early stage. Microscopic Agglutination Test (MAT) which is commonly used in diagnosing leptospirosis able to detect widely leptospiral serovar from the environment. However, the latency of MAT during early onset causes inconclusive diagnosis thus proper treatment towards the patient are delayed. PCR on the other hand uses the bacterial outer membrane proteins (OMPs) as their target region. However, the heterogeneity and variation of the genome causes current widely used LipL32 as the target DNA sequences less efficient in detecting pathogenic and intermediate species of Leptospira. Through knowledge of bioinformatics and analysis, several new target proteins can be developed that are more sensitive in detecting pathogenic and intermediate species of Leptospira. Through bioinformatics analysis, we are able to develop several DNA target sequences which are 16S rRNA, LipL21 and LipL41 in detecting saprophytic, intermediate and pathogenic species of Leptospira respectively. These target proteins are integrated into a TaqMan multiplex assay system. The developed assay is able to amplified Leptospira’s DNA thus able to detect saprophytic, intermediate and pathogenic species as low as 1 ng/µL. The developed TaqMan multiplex assay has the analytical specificity of 100% in detecting Leptospira species with 85% able to differentiate between the Leptospira species. The developed TaqMan multiplex assay also able to detect the pathogenic species upon clinically diagnosed specimens thus it is suitable to be used clinically in diagnosing the disease.

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