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    洋葱伯克霍尔德菌群3种检测方法的比较

    Comparison of Three Detection Methods for Burkholderia Cepacia Complex

      摘要
    • 摘要:
      目的 通过对环介导等温扩增(loop-mediated isothermal amplification，LAMP)、基于聚合酶链式扩增(polymerase chain reaction，PCR)的SYTO 9染料法及TaqMan探针实时荧光定量PCR法(简称TaqMan探针法)3种检测方法的比较，旨在建立一种能够快速、准确检测24株洋葱伯克霍尔德菌群的PCR方法。
      方法 根据 NCBI 数据库中24株洋葱伯克霍尔德菌的分子生物学信息，筛选出洋葱伯克霍尔德菌所特有的多个候选序列片段，设计能同时检出24株洋葱伯克霍尔德菌的特异性引物和探针，同时探索了LAMP法、基于PCR的SYTO 9染料法及Taqman探针法3种检测方法，优化筛选出最佳退火温度，并采用39株实验菌株对洋葱伯克霍尔德菌群检测方法开展了验证研究。
      结果 采用LAMP法无法实现对洋葱伯克霍尔德菌群的有效检出，采用SYTO 9染料法和TaqMan探针法可以实现对20多株洋葱伯克霍尔德菌群的有效检出，而TaqMan探针法扩增效率更高，检测灵敏度、重复性、稳定性更好，能够满足本研究的要求。
      结论 本研究建立了洋葱伯克霍尔德菌群的TaqMan探针快速检测方法，相较于LAMP法和SYTO 9染料法，该方法具有快速、简便和敏感性高等优点，为洋葱伯克霍尔德菌群的快速检测提供了技术支持。

       

      Abstract:
      OBJECTIVE To establish a rapid and accurate PCR method for detecting 24 strains of Burkholderia cepacia complex(Bcc) by comparing three detection methods of loop-mediated isothermal amplification(LAMP), SYTO 9 dye method based on polymerase chain reaction(PCR) and TaqMan probe real-time fluorescent quantitative PCR method( TaqMan probe method).
      METHODS According to the molecular biological information of 24 strains of Bcc in the NCBI database, multiple candidate sequence fragments unique to Bcc were screened out, and specific primer and probe that could simultaneously detect 24 strains of Bcc were designed. At the same time, the detection methods of LAMP, SYTO 9 dye method based on PCR and Taqman probe were explored, and the optimal annealing temperature was optimized and screened. The 39 experimental strains were used to verify the Bcc detection method.
      RESULTS LAMP method could not effectively detect Bcc, SYTO 9 dye method and TaqMan probe method could effectively detect more than 20 strains of Bcc, while TaqMan probe method had higher amplification effect, better detection sensitivity, repeatability and stability, which could meet the requirements of this study.
      CONCLUSION In this study, a TaqMan probe method for rapid detection of Bcc was established. Compared with LAMP method and SYTO 9 dye method, this method has the advantages of fast, simple and high sensitivity, and provides technical support for the rapid detectionof Bcc.

       

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