[Establishment of PCR assays and genetic polymorphism analysis of genes encoding Clostridium perfringens β₂ toxin...

Objective: To establish and optimize PCR methods for the gene encoding of Clostridium perfringens β₂ toxin (cpb) and atypical-cpb (aty-cpb), analyze the epidemiological characteristics and genetic polymorphism of the cpb of Clostridium perfringens in 9 Chinese areas from 2016 to 2021. Methods: The cpb of 188 Clostridium perfringens strains were examined by PCR; the cpb sequences were acquired by whole-genome sequencing to analyze the genetic polymorphism. Using Mega 11 and the Makeblastdb tool, a phylogenetic tree, and cpb-library based on 110 strains carrying the cpb were produced. Using the Blastn technique, a comparison was made to discover sequence similarity between consensus-cpb (con-cpb) and aty-cpb. Results: The specificity of PCR assay for the cpb and aty-cpb was verified. The PCR results for cpb amplification were highly consistent with the whole-genome sequencing approach (Kappa=0.946, P<0.001). A total of 107 strains from nine regions in China carried cpb, 94 types A strains carried aty-cpb, 6 types A strains carried con-cpb, and 7 types F strains carried aty-cpb. The nucleotide sequence similarity between the two coding genes was 68.97%-70.97%, and the similarity between the same coding genes was 98.00%-100.00%. Conclusions: In this study, a specific PCR method for cpb toxin was developed, and the previous PCR method for detecting aty-cpb was improved. aty-cpb is the primary gene encoding of β₂ toxin. There is a significant nucleotide sequence variance between the various cpb genotypes.