Optimized DNA extraction from fecal samples using non-stool kits to improve PCR detection of protozoan parasites: A focus on Blastocystis sp.

PCR-based detection of intestinal protozoa frequently faces challenges due to inadequate DNA recovery or the presence of inhibitors within fecal samples. Therefore, optimizing extraction protocols through pretreatment strategies is crucial to enhance DNA yield and quality, ensuring reliable molecular detection. This study aimed to evaluate whether combining conventional, non-stool-specific extraction kits with pretreatment protocols could enhance DNA yield and purity from fecal samples and enable reliable PCR-based detection of intestinal protozoa, such as Blastocystis sp. Five pretreatment methods based on lysis and the addition of Proteinase K were applied in combination with three commercial DNA extraction kits (FavorPrep™ Viral Nucleic Acid Extraction Kit, Invitrogen PureLink™ Genomic DNA Mini Kit, and IndiMag® Pathogen Kit). DNA yield and purity were assessed from 545 fecal samples, and different protocols were compared using statistical analyses. In addition, PCR amplification was conducted to detect Blastocystis sp. and other protozoan parasites. All pretreatment protocols provided satisfactory DNA yield and purity. However, significant variation was observed among kit-pretreatment combinations, with the Invitrogen-Pretreatment3 combination achieving the highest DNA yield (332.75 ng/μL) and good purity (A260/A280 = 1.69). While all tested extraction protocols enabled successful PCR detection of Blastocystis, differences in DNA quantity and quality were observed, highlighting the impact of pretreatment selection on overall extraction efficiency. Even the lowest-yield combination, IndiMag-Pretreatment2, produced DNA sufficient for reliable protozoan amplification. Non-stool extraction kits, when combined with appropriate pretreatment strategies, can provide DNA of sufficient quantity and quality for PCR-based detection of intestinal protozoa.