Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.
Mol Cell Probes. 2010 Apr;24(2):68-71. doi: 10.1016/j.mcp.2009.10.003. Epub 2009 Oct 13.
A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of Yersinia enterocolitica. The assay used specifically designed primers to target within the phoP gene and correctly identified all 37 strains of Y. enterocolitica and 50 non-Y. enterocolitica strains. The probability of detection was 100%, when the DNA of extracted from 10(1) CFU Y. enterocolitica was used as template in LAMP assay. Prior to the LAMP assay, a sample preparation protocol was applied that included a pre-enrichment step in Luria-Bertani broth, followed by extraction and purification of DNA. In this way, 102 various food samples were investigated for Y. enterocolitica including 79 minced pork samples and 23 powdered milk samples. The accuracy of LAMP was shown to be 100% when compared to the standard method, ISO 10273. This combination of sample enrichment, and LAMP assay can detect 2.2 CFU per 100 g food samples. The overall analysis time for the LAMP assay was approximately 24 h. This is in contrast to 5 days of analysis time required for the traditional culture method. Consequently, the LAMP described here, has the potential to become a standardized method for the rapid detection of Y. enterocolitica in diagnostic laboratories once further validated by inter-laboratory studies.
环介导等温扩增 (LAMP) 法已被开发并验证用于特异性检测小肠结肠炎耶尔森氏菌。该检测方法使用了专门设计的引物，针对 phoP 基因内的靶点，并正确识别了所有 37 株小肠结肠炎耶尔森氏菌和 50 株非小肠结肠炎耶尔森氏菌。当使用 10(1) CFU 小肠结肠炎耶尔森氏菌提取的 DNA 作为模板进行 LAMP 检测时，检测概率为 100%。在进行 LAMP 检测之前，应用了一种样品制备方案，包括在 Luria-Bertani 肉汤中的预富集步骤，然后提取和纯化 DNA。通过这种方式，对包括 79 份肉末样本和 23 份奶粉样本在内的 102 种各种食品样本进行了小肠结肠炎耶尔森氏菌的检测。与标准方法 ISO 10273 相比，LAMP 的准确率为 100%。这种样品富集与 LAMP 检测的结合可以检测到每 100 克食品样本中 2.2 CFU 的小肠结肠炎耶尔森氏菌。LAMP 检测的总分析时间约为 24 小时。相比之下，传统培养方法需要 5 天的分析时间。因此，一旦通过实验室间研究进一步验证，本文所述的 LAMP 具有成为诊断实验室中快速检测小肠结肠炎耶尔森氏菌的标准化方法的潜力。
