Development of optimized DNA extraction protocols for the quantification of Eimeria spp. from chicken feces using real-time PCR

Coccidiosis is one of the most significant parasitic diseases in chickens, caused by protozoan parasites of the genus Eimeria, and leads to substantial economic losses in the poultry industry. The severity of coccidial infection varies greatly depending on the number of ingested sporulated oocysts, highlighting the importance of quantitative analysis; however, no suitable DNA extraction kit has yet been developed to effectively disrupt the robust oocyst wall of Eimeria in fecal samples. This study optimized the DNA extraction methods to improve the sensitivity and reliability of Eimeria spp. detection. Nine commercially available DNA extraction kits were evaluated. All kits were evaluated at an oocyst concentration of 10⁵, and the TANBead Pathogen kit yielded the lowest Ct value (15.67 ± 0.48), indicating the highest extraction performance. This was followed by the TANBead Tissue kit (16.55 ± 0.20), Promega RCS PureFood GMO kit (17.13 ± 0.28), TANBead Viral kit (17.63 ± 0.60), Infusion Tech Soil ∙ Fecal gDNA extraction kit (20.18 ± 1.25), Promega RSC simply RNA Tissue kit (20.56 ± 1.59), QIAGEN Blood & Tissue kit (21.23 ± 0.61), APBio Sporozoa kit (22.05 ± 0.18) and QIAGEN Stool mini kit (23.94 ± 0.61). Among these, three kits (TANBead Pathogen kit, APBio Sporozoa kit, and Infusion Tech Soil ∙ Fecal gDNA kit) were selected for optimization. The TANBead kit was improved its performance by adjusting temperature and incubation time, reducing Ct value from 15.67 to 12.83 at a concentration of 10⁵ oocysts. The APBio kit achieved Ct reduction from 22.05 to 11.06 by modifying bead size and buffer composition, while the Infusion Tech kit improved from 20.18 to 13.66 by optimizing buffer composition and temperature conditions. Moreover, all three optimized kits showed R² values > 0.99 in the standard curve, indicating high linearity and reliability of quantitative analysis. These results demonstrate that the optimized protocols provide an effective and reliable approach for the quantitative detection of Eimeria spp. from fecal samples, contributing to improved monitoring and control of coccidiosis in poultry.