MOLECULAR DETECTION OF PATHOGENIC AND NON-PATHOGENIC LEPTOSPIRA BASED ON MULTIPLEX PCR

Background and Aim:Leptospirosis is a serious zoonotic infection and the most prevalent disease in the tropical and subtropical region such as the north of Iran. The Leptospiral lipl32 gene expressed only in pathogenic Leptospira. 16S rRNA gene sequences are used to identify the species of Leptospira. The aim of this study was to identify a molecular method to identify pathogenic and nonpathogenic Leptospira by Multiplex PCR based on lipl32 and 16s rRNA genes. This method was also able to differentiate between saprophytic and pathogenic leptospires.Methods:Saprophytic and pathogenic Leptospira serovars were used in this study. The bacteria were inoculated into the selective culture medium and extraction of the genomic DNA was performed by the standard Phenol-Chlorophorm method. The specific primers for a proliferation of lipl32 and 16SrRNA genes were used. The specificity and sensitivity of the PCR method were evaluated.Results:The PCR product for the genes of the lipl32 and 16SrRNA was 272bp and 240bp respectively. The sensitivity amplification for the multiplex assay was 10-7 pg for 16S rRNA gene and 10-4 pg for lipl32 gene.Conclusion:In this study by conducting a simple PCR to spend a minimum of time; were identified as pathogenic and non-pathogenic Leptospira serovars. The results showed that a molecular diagnostic test with high specificity and sensitivity suitable as PCR using 16S rRNA and lipl32 primers for distinguishing between pathogenic and nonpathogenic serovars from each other, very efficient.

Keywords:

Leptospirosis , Multiplex PCR , lipl32 , detection , 16s rRNA