张爱佳, 孙启杰, 黄英, 高鹏亚, 刘志楠, 彭筱婧, 刘洋, 徐雪芳, 卓秀伟, 张炜华, 吴长德, 卢选成. 一例婴儿B型肉毒中毒的实验室诊断[J]. 疾病监测, 2020, 35(6): 557-562. DOI: 10.3784/j.issn.1003-9961.2020.06.021
引用本文: 张爱佳, 孙启杰, 黄英, 高鹏亚, 刘志楠, 彭筱婧, 刘洋, 徐雪芳, 卓秀伟, 张炜华, 吴长德, 卢选成. 一例婴儿B型肉毒中毒的实验室诊断[J]. 疾病监测, 2020, 35(6): 557-562. DOI: 10.3784/j.issn.1003-9961.2020.06.021
Aijia Zhang, Qijie Sun, Ying Huang, Pengya Gao, Zhinan Liu, Xiaojing Peng, Yang Liu, Xuefang Xu, Xiuwei Zhuo, Weihua Zhang, Changde Wu, Xuancheng Lu. Laboratory identification of an infant botulism B case[J]. Disease Surveillance, 2020, 35(6): 557-562. DOI: 10.3784/j.issn.1003-9961.2020.06.021
Citation: Aijia Zhang, Qijie Sun, Ying Huang, Pengya Gao, Zhinan Liu, Xiaojing Peng, Yang Liu, Xuefang Xu, Xiuwei Zhuo, Weihua Zhang, Changde Wu, Xuancheng Lu. Laboratory identification of an infant botulism B case[J]. Disease Surveillance, 2020, 35(6): 557-562. DOI: 10.3784/j.issn.1003-9961.2020.06.021

一例婴儿B型肉毒中毒的实验室诊断

Laboratory identification of an infant botulism B case

  • 摘要:
    目的对2019年2月来自北京儿童医院1例疑似肉毒中毒的婴儿粪便样品进行实验室诊断,判定是否为肉毒毒素中毒。
    方法按照GB 4789.12 — 2016稀释处理样品,利用动物实验鉴定肉毒毒素、接种增菌培养基观察变化、采用卵黄培养基进行分离纯化,扩增测序16S rRNA基因并进行序列比对,采用荧光定量PCR方法进行毒素基因分型。
    结果粪便样本稀释后对小鼠进行腹腔注射,小鼠出现呼吸急促、腹式呼吸,并在4~6 h内死亡,呈现肉毒中毒典型症状。 在卵黄培养基平板上分离到典型肉毒梭菌,镜检观察到芽孢形态,测得的16S rRNA基因序列与GenBank中公开的肉毒梭菌16S rRNA基因序列进行比对,相似率>99%,荧光定量PCR检测显示B型毒素基因阳性,判定该婴儿为B型肉毒中毒。
    结论此次实验按照国家标准和本实验室建立的荧光定量PCR方法进行鉴定,检测结果和诊断方案为我国婴儿B型肉毒中毒诊断提供参考,也为分析肉毒中毒地域性提供证据,为临床鉴定婴儿B型肉毒中毒提供参考案例。

     

    Abstract:
    ObjectiveA laboratory test of the stool sample collected from a suspected infant botulism case in Beijing Children’s Hospital in February 2019 was conducted for the final confirmation.
    MethodsThe sample was diluted according to GB 4789.12—2016 for the botulinum toxin identification in animal experiments. The sample was inoculated in enrichment medium to observe its change and inoculated in yolk medium for strain isolation purification. PCR amplification technology was used to amplify 16S rRNA, and the sequences of the target fragments were aligned.
    ResultsMice were injected intraperitoneally with dilution of stool sample, and the mice developed shortness of breath, abdominal breathing and died within 4–6 hours, showing typical symptoms of botulism. The typical Clostridium botulinum was isolated on the yolk medium plate, and the spore morphology was observed by gram staining. The gene sequence alignment showed that the sequence of the 16S rRNA shared 99% similarity with that of C. botulinum. Fluorescent quantitative PCR showed that toxin gene B of C. botulinum was positive, and the infant was diagnosed with botulism B.
    ConclusionThe test results and diagnosis protocol of this study provide reference for the diagnosis and prevention as well as of clinical identification of botulism B in infants in China.

     

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