摘要
为建立一种快速检测艰难梭菌(Clostridiu difficile)两种毒素基因的方法,参照GenBank上发布的艰难梭菌基因组序列,以tcdA和tcdB为靶基因设计特异性引物和TaqMan探针,经过优化引物、探针浓度以及反应条件,建立了可以同时检测两种毒素基因的艰难梭菌双重实时荧光PCR方法。该方法与其他菌株无交叉反应,特异性强;组内和组间试验变异系数均小于5%,具有良好的重复性;对tcdA和tcdB重组质粒的最低检测限分别为9.94和9.63 copies/μL,具有较高的灵敏度。应用本试验建立的方法对180份口岸截获的两栖、爬行类活体动物以及市场售卖宠物的粪便样品进行检测,发现2份宠物犬粪便样品呈阳性,提示该菌在宠物群体中存在传播风险。结果表明,本研究建立的方法适用于出入境检验检疫、医疗、食品、疾控等领域中动物源艰难梭菌的快速检测,为防范该菌危害人类和动物健康提供了技术支撑。
In order to rapidly detect two toxin genes of Clostridium difficile(C.difficile),specific primers and TaqMan probes were designed targeting its tcdA and tcdB genes,based on its genome sequences published in GenBank After optimizing the concentrations of primers and probe as well as reaction conditions,a duplex real-time fluorescent PCR assay for simultaneous detection of the two toxin genes was successfully developed.The established assay failed to crossly react with other bacterial strains,demonstrating strong specificity:both the intra-and inter-assay coefficients of variation(CVs)were less than 5%,indicating good repeatability:the limits of detection(LODs)for the tcdA and tcdB recombinant plasmids were 9.94 and 9.63 copies/μL,respectively,reflecting high sensitivity.180 fecal samples from live amphibians and reptiles intercepted at ports and from pets sold in markets were detected using the established assay,2 positive samples derived from pet dogs were tested positive,indicating a potential risk of C.difficile transmission among pets.In conclusion,the established assay was suitable for rapid detection of animal-derived C.difficile in fields including but not limited to entry-exit inspection and quarantine,healthcare,food safety and disease control,technically supporting future prevention of potential threats that the bacterium may pose to human and animal health.
作者
杜思乐
王素秋
徐浩
闫梦茜
白子龙
张小寒
徐子涵
刘朋朋
高志强
汪琳
蒲静
Du Sile;Wang Suqiu;Xu Hao;Yan Mengxi;Bai Zilong;Zhang Xiaohan;Xu Zihan;Liu Pengpeng;Gao Zhiqiang;Wang Lin;Pu Jing(Science and Technology Research Center of China Customs,Beijing 101113,China;Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs,Shenzhen 518045,Guangdong,China;Capital Normal University,Beijing 100048,China)
出处
《中国动物检疫》
2026年第2期122-129,139,共9页
China Animal Health Inspection
基金
国家重点研发计划项目(2022YFD1800705)
中国海关科学技术研究中心自立课题项目(2023HZ12)。
