黄英, 史芸, 叶长芸, 徐雪芳. 利用实时荧光定量PCR 技术检测毒素基因快速鉴定A/B型肉毒梭菌方法的建立[J]. 疾病监测, 2019, 34(9): 844-848. DOI: 10.3784/j.issn.1003-9961.2019.09.015
引用本文: 黄英, 史芸, 叶长芸, 徐雪芳. 利用实时荧光定量PCR 技术检测毒素基因快速鉴定A/B型肉毒梭菌方法的建立[J]. 疾病监测, 2019, 34(9): 844-848. DOI: 10.3784/j.issn.1003-9961.2019.09.015
Ying Huang, Yun Shi, Changyun Ye, Xuefang Xu. Establishment of real-time PCR assays for rapid detection of Clostridium botulinum type A and B[J]. Disease Surveillance, 2019, 34(9): 844-848. DOI: 10.3784/j.issn.1003-9961.2019.09.015
Citation: Ying Huang, Yun Shi, Changyun Ye, Xuefang Xu. Establishment of real-time PCR assays for rapid detection of Clostridium botulinum type A and B[J]. Disease Surveillance, 2019, 34(9): 844-848. DOI: 10.3784/j.issn.1003-9961.2019.09.015

利用实时荧光定量PCR 技术检测毒素基因快速鉴定A/B型肉毒梭菌方法的建立

Establishment of real-time PCR assays for rapid detection of Clostridium botulinum type A and B

  • 摘要:
    目的建立针对A/B型肉毒梭菌毒素基因的实时荧光定量PCR(real-time PCR)检测方法,构建标准曲线,评价该方法的特异性、敏感性及检测下限,以提高肉毒梭菌检测的快速性、准确性。
    方法根据肉毒梭菌A/B型毒素基因设计特异性引物及探针,优化反应条件,建立real-time PCR反应体系。 用25种细菌评价该方法的特异性,使用含有A/B型毒素基因特异性序列的重组质粒标准品构建标准曲线,模拟粪便标本,评价建立方法的灵敏度。
    结果建立的real-time PCR检测方法特异性好,携带A/B型毒素基因的重组质粒均出现相应特异性扩增曲线,对其他25种细菌进行试验扩增时,均未见出现特异性扩增曲线。 根据重组质粒标准品构建的标准曲线可确定其对肉毒梭菌A/B型毒素基因检测灵敏度分别为5.04×102拷贝/μl和6.91×102拷贝/μl。 模拟粪便标本中含有目的基因重组质粒的检测下限为A型1.71×103拷贝/μl,B型2.14×103拷贝/μl。
    结论本研究设计了适合检测国内肉毒梭菌 A/B 型的引物和探针,建立 real-time PCR 快速检测体系,为我国肉毒梭菌 A/B 型菌株的快速鉴定提供一种新的技术手段。

     

    Abstract:
    ObjectiveTo establish real-time PCR assays for the toxin gene detections of Clostridium botulinum type A and B, construct standard curves and evaluate the specificities, sensitivities and detection thresholds of the assays, and provide evidence for the rapid and accurate detection of C. botulinum.
    MethodsSpecific primers and probes were designed based on the sequences of toxin genes of C. botulinum type A and B. Real-time PCR assays were established with optimized reaction conditions. 25 other intestinal bacteria and common bacteria were used to test the specificities of the assays. Standard curve construction, fecal sample simulation and sensitivity measurement were achieved with recombinant plasmids containing toxin genes of C. botulinum type A and B.
    ResultsThe specificities of the real-time PCR assays were high. Specific amplification curves were observed in recombinant plasmids containing toxin genes of C. botulinum type A and B. No specific amplifications were found for the 25 other bacteria. The detection thresholds of toxin genes of C. botulinum type A and type B were 5.04×102 copy/μl and 6.91×102 copy/μl respectively according to the amplification curves. The detection thresholds of recombinant plasmids containing toxin genes of C. botulinum type A and B in artificial fecal samples were 1.71×103 copy/μl and 2.14×103 copy/μl respectively.
    ConclusionIn this study, real -time PCR assays for the toxin gens detections of C. botulinum type A and type B of China were established, which can be applied in the rapid detection of C. botulinum.

     

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