Comparison of two methods for DNA extraction from human inoculated feces with S almonella sp . for qPCR amplification.

Introduction: The Polymerase Chain Reaction (PCR) has been applied to detect challenging-to-isolate pathogenic microorganisms from food or clinical samples. However, inhibitors or inherent elements of the samples can impact its efficiency. The quantity and quality of extracted DNA are crucial. Objective: To compare two extraction methods for obtaining PCR-amplifiable DNA in real-time PCR (qPCR). Methodology: DNA was extracted using the Phenol-Chloroform-Isoamyl Alcohol (PC) and Chelex® 100 (Chlx) methods from stool samples inoculated with Salmonella spp. previously cultured in selective Selenite Cystine (SC) and Tetrationate (T) broths. Results: DNA was quantified by spectrophotometry and amplified by qPCR to detect Salmonella spp. The average DNA purity value using the PC method from SC culture was 1.61 ± 0.21, while from T culture it was 1.56 ± 0.21. The Chlx method from SC culture resulted in a purity of 1.57 ± 0.24, while from T culture it was 1.79 ± 0.20. However, better amplification was achieved using the PC extraction method from SC culture. Conclusions: It was determined that the PC extraction method from SC culture provides PCR-amplifiable DNA in Real-Time PCR, and pre-PCR culturing is advantageous as it enhances the bacterial target and dilutes inhibitors.