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Background: Leptospirosis is a worldwide zoonotic disease, which is still often misdiagnosed. Laboratory detection of Leptospira is challenging since the bacteraemia is quite short for molecular detection, however, the rise of the antibody is late to post the infection. Urine can be a potential alternative sample for PCR detection in leptospirosis. The PCR method requires a reliable DNA template, which is obtained from good DNA extracting methods. The study aimed to determine the best method of extraction Leptospira DNA from the urine sample, as well as evaluating the effect of time storage and temperature for its DNA stability. Methods: This study was utilizing three different DNA isolation methods; silica based with spin column, spin column chromatography using resin as separation matrix, and solution method with guanidine isothiocyanate. The yields were examined for its concentration and purity. Leptospira’s SecY gene was detected with realtime PCR. The influences of storage temperature and the life time of the DNA were also studied. Results: The yield of DNA isolation using resin showed the highest concentration (7.94+2.11 μg/mL) and highest Leptospira DNA amplification copy number (50167.92+1.19). Storage temperature at 4°C, -20°C, and -80°C and life time of 91 days did not have any effect on the quality and quantity of Leptospira DNA isolated from spiked urine. Conclusions: DNA isolation using spin column chromatography with resin as separation matrix has the best quality and quantity based on the purity and concentration of DNA and the higher number of amplified SecY gene. (Health Science Journal of Indonesia 2020;11(2):77-84)

Comparison of DNA extraction methods for molecular identification of pathogenic Leptospira in the urine samples