Optimizing DNA extraction methods for enhanced detection of pathogenic Leptospira spp. in cultures using PCR

Abstract

Leptospirosis is a worldwide zoonosis caused by Leptospira interrogans. Laboratory diagnosis of leptospirosis can be done through sample culture. However, microscopic observation of positive Leptospira spp. culture need to be supported by molecular detection to confirm the presence of pathogenic Leptospira spp. This study aimed to compare the effectiveness of two DNA extraction kits, from QIAGEN and Zymo Research, to extract bacterial DNA genome from positive Leptospira spp. culture for downstream molecular detection. This study is an analytical observational study with a cross-sectional design conducted in June-September 2023, and part of the PESTO-RITA 2023 study. Fourteen identified positive Leptospira spp. culture, with Leptospira spp. movement under dark field microscopy, was used for DNA extraction using both extraction kits. QIAGEN kit yielded higher mean DNA concentration (0.900±0.161 vs 0.790±0.167 log10 µg/mL, p>0.05) and better DNA purity (1.854 vs 1.632, p<0.05) than the Zymo Research kit. PCR results, targeting the lipL32 gene, showed that two samples extracted with QIAGEN and one sample extracted with Zymo Research were positive for pathogenic Leptospira spp. Our preliminary results showed that QIAGEN kit yielded better DNA concentration and purity from positive Leptospira spp. culture.

Keywords

• dna concentration
• dna extraction
• dna purity
• leptospira detection
• pcr