可视化和实时荧光环介导恒温扩增技术快速检测原料乳中的沙门氏菌

Rapid detection of Salmonella in raw milk by visual and real-time fluorescence loop-mediated isothermal amplification-based methods

摘要:

【背景】原料乳质量控制是乳制品生产流程中的关键环节，沙门氏菌是污染原料乳的主要致病菌之一。随着生物检测技术的不断发展，建立用于快速检测原料乳中沙门氏菌的实用型方法具有重要意义。【目的】评价可视化环介导恒温扩增技术(visual loop-mediated isothermal amplification，V-LAMP)和实时荧光环介导恒温扩增技术(real-time fluorescence loop-mediated isothermal amplification，RF-LAMP)方法快速检测原料乳中沙门氏菌的实效性。【方法】建立沙门氏菌LAMP反应体系和反应条件，对比V-LAMP和RF-LAMP两种方法的特异性、灵敏度、稳定性及其应用于实际样品检测的效用。【结果】采用V-LAMP和RF-LAMP方法检测沙门氏菌标准菌株，其结果均为阳性，检测非沙门氏菌标准菌株其结果均为阴性；该两种方法的检出限为13 CFU/mL，检测重复率均为100%；经检测10份人工污染样品，该两种方法的检测结果与实际结果高度一致；经检测73份实际样品，相对于标准方法，该两种方法其相对特异性分别为94.12%和92.65%，相对敏感度分别为100%和100%，相对准确率分别为94.52%和93.15%，一致性χ2值分别为2.25和3.20，与标准方法无明显差异(P>0.05)。【结论】V-LAMP和RF-LAMP方法检测特异性好、灵敏度高、稳定性好，适用于原料乳中沙门氏菌的快速检测。

Abstract:

[Background] Quality control of raw milk is a key part in the production process of dairy products. Salmonella is one of the main pathogenic bacteria that can contaminate raw milk. With the development of biological detection technology, it is of great significance to establish a practical method for rapid detection of Salmonella in raw milk. [Objective] To evaluate practical application effect of rapid Salmonella detection in raw milk by visual loop-mediated isothermal amplification (V-LAMP) and real-time fluorescence loop-mediated isothermal amplification (RF-LAMP), respectively. [Methods] We established the LAMP reaction system and optimized the reaction conditions, then tested and compared the specificity, sensitivity, stability and effectiveness in practical samples detection by using V-LAMP and RF-LAMP methods, respectively. [Results] Through the rapid detection of V-LAMP and RF-LAMP, all Salmonella standard strains were detected positive, while all non-Salmonella standard strains were negative. The limit of detection for the two methods was calculated to 13 CFU/mL. The detection accordance rate of the two methods were both 100%. The results obtained by detecting Salmonella in 10 artificial contaminated samples were highly consistent with the actual results. With respect to the result obtained by conventional detection method in the 73 real samples, the relative specificity of the two methods were respectively 94.12% and 92.65%, the relative sensitivity were respectively 100% and 100%, the relative accuracy were respectively 94.52% and 93.15%, and the χ2 value for consistency analysis were respectively 2.25 and 3.20, showing that no significant difference was found in the results above by the three mentioned methods (P>0.05). [Conclusion] V-LAMP and RF-LAMP methods show good specificity, high sensitivity, and high consistency, which makes them applicable for rapid detection of Salmonella in raw milk.