Application of polymerase chain reaction (PCR) methods in distinguishing pathogenic and saprophytic Leptospira from the traditional market environment in Denpasar City

Abstract

Background: Leptospirosis disease is still a health problem in Indonesia. The number of leptospirosis cases and deaths in Indonesia tends to increase. Indirect transmission of Leptospira bacteria can occur through poor environmental sanitation polluted by rat urine has the potential to cause leptospirosis. Traditional markets are good places for rats to breed. The purpose of this study was to detect the presence of contamination by pathogenic and saprophytic Leptospira bacteria in the environment around traditional markets in Denpasar City by polymerase chain reaction (PCR) method using three specific primers. Methods: The sample is water from the market environment, taken at 19 location points from eight traditional markets in Denpasar City. After being homogenized, the sample water is filtered. Isolation of specific genes from samples by the PCR method was performed to differentiate pathogenic and saprophytic leptospira using three specific primers designed from the 16S rRNA gene. Starting from the DNA extraction stage, amplification by PCR, and detection of PCR product DNA by electrophoresis. Results: This study found that from 19 water sampling locations, 5/19 (26.3%) point locations found specific DNA genes for pathogenic Leptospira bacteria. 10/19 (52.6%) point locations found specific genes for saprophytic Leptospira bacteria DNA, and 4/19 (21.1%) point locations found no specific genes for pathogenic or saprophytic Leptospira bacteria. Conclusion: PCR examination using a combination of three primers could distinguish the presence of pathogenic Leptospira, saprophytic Leptospira, and the absence of pathogenic and saprophytic Leptospira simultaneously from traditional market environments in Denpasar City.