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不同来源产气荚膜梭菌β_(2)毒素编码基因的PCR检测方法建立及遗传多态性分析

Establishment of PCR assays and genetic polymorphism analysis of genes encoding Clostridium perfringensβ_(2)toxin from different sources
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摘要 目的建立并优化产气荚膜梭菌β_(2)毒素编码基因(cpb2)和非典型cpb(2 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cpb(2 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。 Objective To establish and optimize PCR methods for the gene encoding of Clostridium perfringensβ_(2) 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 ofβ_(2) toxin.There is a significant nucleotide sequence variance between the various cpb2 genotypes.
作者 郑浩然 王媛媛 白璐璐 钟佳鑫 卢金星 吴媛 邓慧玲 Zheng Haoran;Wang Yuanyuan;Bai Lulu;Zhong Jiaxin;Lu Jinxing;Wu Yuan;Deng Huiling(Shaanxi University of Traditional Chinese Medicine,Xi'an 712046,China;National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Xi'an Central Hospital,Xi'an 710003,China)
出处 《中华流行病学杂志》 CAS CSCD 北大核心 2023年第4期636-642,共7页 Chinese Journal of Epidemiology
基金 国家重点研发计划(2021YFC2301000) 传染病预防控制国家重点实验室自主研究课题(2021SKLID207)。
关键词 产气荚膜梭菌 β_(2)毒素 遗传多态性 Clostridium perfringens β_(2)toxin Genetic polymorphism
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