Cheng Jinxia, Zeng Jing, Liu Li, Wei Haiyan, Zhao Xiaojuan, Zhang Ximeng, Zhang Lei, Zhang Haiyu
College of Food Science and Engineering, Beijing University of Agriculture, Beijing 102206, China.
Email:
Zhonghua Yu Fang Yi Xue Za Zhi. 2014 Feb;48(2):133-7.
A novel method of Nano-Immunomagnetic Separation (Nano-IMS) plus Real-time PCR was established for detecting Vibrio cholerae.
The Nano-Immunomagnetic Beads were created by using the monoclonal antibody of Vibrio cholerae, which was named Nano-IMB-Vc. Nano-IMB-Vc has specific adsorption of Vibrio cholerae, combined with Real-time PCR technology, a method for rapid detection of Vibrio cholerae was established. The capture specificity of Nano-IMB-Vc was tested by using 15 bacteria strains. The specificity of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria strains. The sensitivity of Nano-IMS plus Real-time PCR were tested in pure culture and in artificial samples and compared with NMKL No.156.
The capture ratio of Nano-IMB-Vc was reached 70.2% at the level of 10(3) CFU/ml. In pure culture, the sensitivity of Nano-IMS plus Real-time PCR was reached at 5.4×10(2) CFU/ml. The specific of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria. The results showed that 102 strains of Vibrio cholerae test results were all positive, and the rest of the 101 strains of non-target bacteria test results were negative. No cross-reaction was founded. Add 1 CFU vibrio cholerae per 25 g sample, it could be detect with Nano-IMS plus Real-time PCR method after 8 hours enrichment.
The Nano-IMS plus Real-time PCR method of Vibrio cholerae established in this study has good specificity and sensitivity, which could be applied to the rapid detection of Vibrio cholerae.
建立一种纳米免疫磁分离（Nano-IMS）结合实时荧光定量聚合酶链反应（Real-time PCR）检测霍乱弧菌的新方法。
利用霍乱弧菌单克隆抗体制备纳米免疫磁珠，命名为纳米免疫磁珠-Vc（Nano-IMB-Vc）。Nano-IMB-Vc对霍乱弧菌具有特异性吸附作用，结合实时荧光定量PCR技术，建立一种快速检测霍乱弧菌的方法。采用15株细菌测试Nano-IMB-Vc的捕获特异性。采用102株目标菌和101株非目标菌测试实时荧光定量PCR方法的特异性。在纯培养物和人工样本中测试Nano-IMS结合实时荧光定量PCR的灵敏度，并与NMKL No.156进行比较。
在10³CFU/ml水平下，Nano-IMB-Vc的捕获率达到70.2%。在纯培养物中，Nano-IMS结合实时荧光定量PCR的灵敏度达到5.4×10²CFU/ml。采用102株目标菌和101株非目标菌测试实时荧光定量PCR方法的特异性。结果显示，102株霍乱弧菌检测结果均为阳性，其余101株非目标菌检测结果均为阴性，未发现交叉反应。每25 g样本中添加1 CFU霍乱弧菌，富集8小时后采用Nano-IMS结合实时荧光定量PCR方法可检测到。
本研究建立的Nano-IMS结合实时荧光定量PCR检测霍乱弧菌方法具有良好的特异性和灵敏度，可应用于霍乱弧菌的快速检测。
