实时荧光环介导等温扩增技术检测牛乳中的蜡样芽孢杆菌

建立牛乳中蜡样芽孢杆菌便捷可靠的检测方法,根据已公布的蜡样芽孢杆菌hbl A基因序列设计内外引物,并向反应体系中加入荧光染料SYBRGreenⅠ,利用实时荧光监测仪,建立实时荧光环介导等温扩增技术(real-time fluorescence loop-mediated isothermal amplification,Real Amp)检测蜡样芽孢杆菌的方法,扩增产物经电泳和酶切鉴定。通过21株致病菌验证RealAmp特异性,并比较了Real Amp与普通环介导等温扩增技术的敏感性,对人工污染的检出限进行了测定。结果表明对21 株致病菌进行特异性实验,4株蜡样芽胞杆菌呈阳性结果,17株非蜡样芽胞杆菌均呈阴性结果。RealAmp检测纯菌的灵敏度为8.2 CFU/m L,比普通环介导等温扩增技术的灵敏度高10倍,人工污染牛乳RealAmp的检出限为8.2 CFU/m L。并且在20 min左右即可判定结果。该方法快速、准确、灵敏度高、操作便捷、可实时监控检测蜡样芽孢杆菌,有望成为快速检测蜡样芽孢杆菌的有效方法。

Detection of Bacillus cereus in Milk by Real-Time Fluorescence Loop-Mediated Isothermal Amplification Method

This study aimed to establish a real-time fluorescence loop-mediated isothermal amplification assay to detect
Bacillus cereus. Based on the published Bacillus cereus hblA gene sequence, primers were designed, and the intercalating
dye SYBR-Green I was added to the reaction system, which allowed the reaction products to be measured by ESE-Quant
Tube Scanner. The products were identified by restriction digestion followed by electrophoresis. The performance of the
assay was evaluated with respect to specificity and sensitivity in comparison with the ordinary LAMP, and the limit of
detection (LOD) for artificially contaminated milk was tested. Among 21 strains tested, four Bacillus cereus strains were
identified, the other 17 strains were negative. The sensitivity for Bacillus cereus was 8.2 CFU/mL in pure cultures, which
was 10 times more sensitive than the ordinary LAMP. The LOD for artificially contaminated milk was 8.2 CFU/mL. The
result could be obtained in 20 minutes. This method has high specificity and sensitivity and is less time-consuming, requiring
only simple equipment and allowing real-time monitoring. It is expected to be applied as an effective method for rapid
detection of B. cereus.