Optimization of a loop-mediated isothermal amplification (lamp) assay for the rapid detection of Clostridium perfringens

Abstract

Summary: This study developed and optimized a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, and specific detection of Clostridium perfringens (C. perfringens), a key pathogen in necrotic enteritis in poultry, causing significant global economic losses. We designed two sets of LAMP primers, ID7 and ID22, targeting the cpa gene of C. perfringens. The CP-LAMP assays were optimized for temperature, time, and primer concentration. We conducted inclusivity tests on 55 C. perfringens strains and exclusivity tests on six other Clostridium species strains and 33 non-Clostridium strains (Escherichia coli, Campylobacter, and Salmonella). Sensitivity was evaluated using serially diluted genomic DNA from C. perfringens ATCC 13124 and compared to PCR. The optimal primer concentrations were 8 μM F3/B3 and 48 μM FIP/BIP. Reactions were performed at 63°C for 30 min. Both primer sets achieved 100 % inclusivity and 88.33 % and 100 % exclusivity for other Clostridium and non-Clostridium isolates, respectively. The CP-LAMP assay's sensitivity and specificity were comparable to PCR, enhancing surveillance and management of necrotic enteritis in poultry farms, potentially reducing its economic impact.

Keywords

• Clostridium perfringens
• Necrotic enteritis
• Rapid detection
• CP-LAMP assay
• PCR