可视化环介导等温扩增法检测牛奶中产志贺毒素大肠埃希氏菌

Determination of Shiga toxin-producing Escherichia coli in milk by visual loop mediated isothermal amplification

中文关键词:  产志贺毒素大肠埃希氏菌  环介导等温扩增  可视化  stx1/2基因

英文关键词:Shiga toxin-producing Escherichia coli  loop mediated isothermal amplification  visualization  stx1/2 gene

中文摘要:

      目的 建立可视化环介导等温扩增(loop mediated isothermal amplification, LAMP)法检测牛奶中产志贺毒素大肠埃希氏菌(Shiga toxin-producing Escherichia coli, STEC)。方法 以stx1/2基因作为检测靶基因, 利用羟基萘酚蓝(hydroxy naphthol blue, HNB)指示剂, 建立一种可视化检测STEC的LAMP方法。同时, 将建立好的LAMP方法与聚合酶链式反应(polymerase chain reaction, PCR)检测方法相比较, 并对人工污染STEC的脱脂乳进行检测。结果 建立的可视化LAMP方法在64℃反应50 min后可凭肉眼观察(紫罗兰色到天蓝色)判定结果。该方法特异性良好, 与大肠埃希菌、沙门氏菌、金黄色葡萄球菌等均无交叉反应, 可以准确区分STEC与非STEC菌。Stx1-LAMP和stx2-LAMP的检出限分别为3.54×10?4和3.54×10?5 ng/μL, 而常规PCR中stx1和stx2的检出限分别为3.54×10?3和3.54×10?2 ng/μL, 即stx1-LAMP检测方法的灵敏度是stx1-PCR的10倍, stx2-LAMP检测方法的灵敏度是stx2-PCR的1000倍。对人工污染STEC菌牛奶样品进行检测, stx1-LAMP和stx2-LAMP分别可检测到细菌浓度为103和102 CFU/mL的加标样品。结论 本研究为STEC菌株提供了一种特异性强、灵敏度高、成本低, 适用于现场和基层检测的可视化LAMP检测方法。

英文摘要:

      Objective To establish a visual loop mediated isothermal amplification (LAMP) method for the detection of Shiga toxin-producing Escherichia coli (STEC) in milk. Methods A LAMP method for visual detection of STEC was established using stx1/2 gene as the target gene and hydroxy naphthol blue (HNB) indicator. At the same time, the established LAMP method was compared with the polymerase chain reaction (PCR) detection method, and the sterilized milk artificially contaminated with STEC were detected. Results The established visual LAMP method could judge the results directly by visual observation (violet to sky blue) after 50 min of reaction at 64℃. The method had good specificity and no cross-reaction with Escherichia coli, Salmonella, Staphylococcus aureus, etc., and could accurately distinguish STEC from non-STEC bacteria. The limits of detection of stx1-LAMP and stx2-LAMP detection methods were 3.54×10?4 and 3.54×10?5 ng/μL, respectively, while the limits of detection of stx1-PCR and stx2-PCR were 3.54×10?3 and 3.54×10?2 ng/μL, respectively. The sensitivity of stx1-LAMP detection method was 10 times that of stx1-PCR, and the sensitivity of stx2-LAMP detection method was 1000 times that of stx2-PCR. For artificially contaminated STEC milk samples, stx1-LAMP and stx2-LAMP could detect the labeled samples with bacterial concentration of 103 and 102 CFU/mL, respectively. Conclusion This study provides a visual LAMP detection method for STEC strains with strong specificity, high sensitivity, and low cost, which is suitable for field and grass-roots detection.