Evaluation of different DNA extraction methods for the detection of Clostridium perfringens by loop-mediated isothermal amplification (LAMP) assay

Abstract

Clostridium perfringens (C. perfringens) is a gram-positive bacterium that causes necrotic enteritis in poultry, resulting in significant economic losses. The development of a reliable and cost-effective diagnostic assay is crucial to prevent the spread of this bacterium. The loop-mediated isothermal amplification (LAMP) assay has emerged as a promising tool for in-field detection of C. perfringens. However, for effective implementation in low-resource settings, a simplified, low-cost DNA extraction method that minimizes equipment needs is crucial. This study aimed to identify an optimal DNA extraction method for the LAMP assay targeting C. perfringens detection. We evaluated four DNA extraction methods, Spin-column (SC), Magnetic Beads (MB), Dipstick (DS), and Hotshot (HS), based on DNA yield, purity, quality, and their compatibility with LAMP and PCR assays. The effectiveness of these methods was tested using spiked broiler feces with reference C. perfringens. Our findings indicated that SC and MB methods yielded DNA of higher purity and quality. Specifically, the SC method demonstrated superior performance in both LAMP and PCR assays across all tested C. perfringens strains. While the HS method was not the top performer, it proved to be the most practical for DNA extraction in resource-limited environments. Sensitivity tests for the LAMP assay showed the highest detection capability with SC-extracted DNA, while DS and HS methods exhibited lower sensitivity. In summary, this study provides valuable information for establishing a practical and adaptable diagnostic approach for C. perfringens detection in low-resource settings, with the HS-DNA extraction method as a feasible option for on-site LAMP assays.