Abstract: The aim of this study was to establish a duplex PCR and a duplex nano-PCR method for simultaneous detection of Eimeria necatrix and Clostridium perfringens. Primers for target gene loci were designed and screened based on the E. necatrix ITS-2 gene and the C. perfringens α toxin gene, respectively, and specific duplex PCR and nano-PCR detection methods for two pathogens were established by optimizing reaction systems and annealing temperatures. PCR amplifications of six other protozoan parasites and three other bacteria were used to verify their specificities. The recombinant plasmids pMD19-T-ITS2 and pMD19-T-CPA were further constructed and diluted into different concentration gradients with 10 times ratio for sensitivity tests of the proposed methods. The results showed that the target fragments of E. necatrix and C. perfringens were about 150 bp and 400 bp, respectively. The PCR amplifications using the two detection methods were negative for other six protozoan parasites and three other bacteria. The minimum detectable limits for E. necatrix and C. perfringens by using dual PCR were 181 copies and 1 050 copies, respectively, while the minimum detectable limits for E. necatrix and C. perfringens by using dual nano-PCR were 1.81 and 105 copies, respectively. Clinical detection showed that the detection results of two established methods were consistent with clinical pathogenic detection. The present study successfully established duplex PCR and duplex nano-PCR methods for detection of E. necatrix and C. Perfringens, with high sensitivity and specificity, providing technical supports for clinical detection of E. necatrix and C. perfringens infections.

Key words: Eimeria necatrix, Clostridium perfringens, PCR, nano-PCR