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

Objective: To establish and optimize PCR methods for the gene encoding of Clostridium perfringens β2 toxin (cpb2) and atypical-cpb2 (aty-cpb2), analyze the epidemiological characteristics and genetic polymorphism of the cpb2 of Clostridium perfringens in 9 Chinese areas from 2016 to 2021. Methods: The cpb2 of 188 Clostridium perfringens strains were examined by PCR; the cpb2 sequences were acquired by whole-genome sequencing to analyze the genetic polymorphism. Using Mega 11 and the Makeblastdb tool, a phylogenetic tree, and cpb2-library based on 110 strains carrying the cpb2 were produced. Using the Blastn technique, a comparison was made to discover sequence similarity between consensus-cpb2 (con-cpb2) and aty-cpb2. Results: The specificity of PCR assay for the cpb2 and aty-cpb2 was verified. The PCR results for cpb2 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 cpb2, 94 types A strains carried aty-cpb2, 6 types A strains carried con-cpb2, and 7 types F strains carried aty-cpb2. 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 cpb2 toxin was developed, and the previous PCR method for detecting aty-cpb2 was improved. aty-cpb2 is the primary gene encoding of β2 toxin. There is a significant nucleotide sequence variance between the various cpb2 genotypes.目的： 建立并优化产气荚膜梭菌β2毒素编码基因（cpb2）和非典型cpb2（aty-cpb2）的PCR检测方法，分析2016-2021年中国9个地区cpb2流行特征和遗传多态性。 方法： 使用PCR方法对188株产气荚膜梭菌菌株的cpb2进行检测，通过全基因组测序获取cpb2序列以分析其遗传多态性，使用Mega 11、Makeblastdb软件对110株产气荚膜梭菌所携带的cpb2构建系统发育树并建库，通过Blastn算法比对后得出cpb2两种不同基因型，即共有cpb2（con-cpb2）和aty-cpb2之间的序列相似性。 结果： 针对产气荚膜梭菌cpb2和aty-cpb2建立及改进的PCR检测方法的特异性好。cpb2的PCR检测结果与全基因组测序结果高度一致（Kappa=0.946，P<0.001）。来自中国9个地区的菌株中共有107株菌携带cpb2，94株A型菌株携带aty-cpb2，6株A型菌株携带con-cpb2，7株F型菌株携带aty-cpb2。两种不同基因型的cpb2核苷酸序列相似性为68.97%~70.97%，相同基因型的cpb2核苷酸序列相似性为98.00%~100.00%。 结论： 本研究建立了特异性好、一致性高的cpb2的PCR检测方法，并针对既往检测aty-cpb2的PCR方法特异性差的缺陷进行了改进。cpb2以aty-cpb2为主且cpb2的不同基因型之间核苷酸序列差异大。.