Natali Moreno1, Piedad Agudelo-Flórez. 1. Grupo de Investigación en Medicina Tropical, Instituto Colombiano de Medicina Tropical, Universidad CES, Medellín, Colombia.
Abstract
UNLABELLED: Serological identification of Leptospira ssp isolates is difficult to achieve. Thus, molecular testing may be of great interest thanks to its high discrimination power, reproducibility and easy interpretation. OBJECTIVE: To implement and validate conventional and multiplex PCR methods (using primers directed against lipl32 and secY/flaB genes, respectively). To assess the capacity of PCR methods to identify pathogenic and saprophytic species of Leptospira ssp. MATERIAL AND METHODS: 22 international reference strains and 12 colombian isolates were used. DNA was extracted with a commercial kit (Wizard). Specificity and sensitivity of both PCR methods were evaluated. RESULTS: The maximum dilution of DNA samples allowing the detection of Leptospira ssp was determined to be 1:10000 for the PCR lipL32 and 1:100/1:1000 for the multiplex PCR secY/flaB. Both PCR didn't detect DNA from microorganisms unrelated to Leptospira ssp. The lipL32 PCR specifically amplified a 423 bp fragment from all pathogenic Leptospira reference strains, while the secY/flaB PCR amplified both 285 bp (secY) and 793 bp (flaB) fragments from 18 reference strains. The lipL32 PCR detected 7/12 colombian isolates, while secY/flaB PCR detected both secY and flaB genes from 6/12 isolates. CONCLUSIONS: Best results were obtained with the lipL32 PCR, which displayed a better sensitivity and a better capacity to detect different strains than the multiplex PCR. The secY primers showed a poor specificity to pathogenic species and a poor sensitivity. Thus, lipL32 primers show high potential for molecular diagnosis of Leptospira spp in clinical and environmental samples.
UNLABELLED: Serological identification of Leptospira ssp isolates is difficult to achieve. Thus, molecular testing may be of great interest thanks to its high discrimination power, reproducibility and easy interpretation. OBJECTIVE: To implement and validate conventional and multiplex PCR methods (using primers directed against lipl32 and secY/flaB genes, respectively). To assess the capacity of PCR methods to identify pathogenic and saprophytic species of Leptospira ssp. MATERIAL AND METHODS: 22 international reference strains and 12 colombian isolates were used. DNA was extracted with a commercial kit (Wizard). Specificity and sensitivity of both PCR methods were evaluated. RESULTS: The maximum dilution of DNA samples allowing the detection of Leptospira ssp was determined to be 1:10000 for the PCR lipL32 and 1:100/1:1000 for the multiplex PCR secY/flaB. Both PCR didn't detect DNA from microorganisms unrelated to Leptospira ssp. The lipL32 PCR specifically amplified a 423 bp fragment from all pathogenic Leptospira reference strains, while the secY/flaB PCR amplified both 285 bp (secY) and 793 bp (flaB) fragments from 18 reference strains. The lipL32 PCR detected 7/12 colombian isolates, while secY/flaB PCR detected both secY and flaB genes from 6/12 isolates. CONCLUSIONS: Best results were obtained with the lipL32 PCR, which displayed a better sensitivity and a better capacity to detect different strains than the multiplex PCR. The secY primers showed a poor specificity to pathogenic species and a poor sensitivity. Thus, lipL32 primers show high potential for molecular diagnosis of Leptospira spp in clinical and environmental samples.