Application of a Multiplex PCR Assay for Molecular Identification of Pathogenic and Non-Pathogenic Leptospires based on lipL32 and 16S rRNA Genes

Abstract

Leptospirosis is a serious zoonotic infection and the most prevalence disease is in the tropical and subtropical region. The definitive diagnosis of Leptospirosis, caused by spirochetes of the genus Leptospira infection is already using culture methods, serological tests such as the microscopic agglutination test (MAT) and molecular detection methods (PCR) are possible. In this study, we used multiplex PCR method for detection of pathogenic and non - pathogenic Leptospira based on lipL32 and 16S rRNA genes. All serovars were obtained from the Leptospira Reference Laboratory of Microbiology Department, Razi Vaccine and Serum Research Institute, Karaj, Iran. The PCR product for the lipL32 and 16S rRNA genes was 272 bp and 240 bp respectively. The sensitivity amplification for the multiplex assay was 10^-6 pg / μl for 16S rRNA gene and 10^-4 pg / μl for lipL32 gene. The sensitivity for multiplex PCR was 10^-3 pg / μl. The results supported the idea that multiplex PCR can be used to detect Leptospira samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much easily than conventional methodologies. Due to the slow growth of Leptospira and the importance of time in diagnosis, molecular methods such as PCR are suggested.

Keywords: 16S rRNA gene; Molecular Identification; Multiplex PCR; lipL32 gene; Leptospira.

Figure 1

Agarose gel electrophoresis of multiplex-PCR products showing the detection of pathogenic and non-pathogenic leptospires. Lane 1:100 bp DNA ladder; Lan 2: non-pathogenic Leptospira (L.biflexa Serovar Semaranga RTCC 2819); Lan 3: non-pathogenic Leptospira (L.biflexa Serovar Semaranga RTCC 2828); Lane 4: pathogenic Leptospira (L.interrogans Serovar Canicola RTCC 2805); Lan 5: pathogenic Leptospira (L.interrogans Serovar Grippotyphosa RTCC 2808); Lan 6: Salmonella enteritidis (RTCC 1621)