摘要
目的 采用纳米免疫磁珠（Nano-IMS）分离霍乱弧菌,建立霍乱弧菌实时荧光PCR（Real-time PCR）检测方法.方法 采用霍乱弧菌单克隆抗体,制备霍乱弧菌纳米免疫磁珠（Nano-IMB-Vc）,特异性吸附霍乱弧菌,结合Real-time PCR技术,建立霍乱弧菌快速检测方法.选用15株典型菌株,对Nano-IMB-Vc捕获特异性进行验证;选取102株霍乱弧菌和101株非目标菌,对所建立的Real-time PCR方法进行特异性验证;采用纯菌的灵敏度检测和基质添加实验对所建立方法的灵敏度进行检测,并与食品中霍乱弧菌的检测方法（NMKL No.156）进行比较.结果 Nano-IMB-Vc在菌体浓度为103 CFU/ml水平,对霍乱弧菌的捕获率最高可达70.2％.Nano-IMB-Vc分离结合Real-time PCR技术,在纯培养、无需增菌情况下,检测灵敏度达到5.4×102 CFU/ml;通过102株霍乱弧菌和101株非目标菌的测试,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,无交叉反应.采用Nano-IMB-Vc分离结合Real-time PCR技术,在食品基质添加试验中,每25 g样品中添加1CFU霍乱弧菌,增菌8h即可检出.结论 Nano-IMB-Vc分离结合Real-time PCR技术,具有良好的特异性,较高的检测灵敏度,适用于霍乱弧菌的快速筛选.
Objective A noval method of Nano-Immunomagnetic Separation （Nano-IMS） plus Real-time PCR was established for detecting Vibrio choleraes.Methods The Nano-Immunomagnetic Beads were created by using the monoclonal antibody of Vibrio choleraes,which was named Nano-IMB-Vc.Nano-IMB-Vc has specific adsorption of Vibrio choleraes,combined with Real-time PCR technology,a method for rapid detection of Vibrio choleraes 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 l01 non-targets bacteria strains.The sensitivity of Nano-IMS plus Real-time PCR were tested in pure culture and in artficial samples and compared with NMKL No.156.Results The capture ratio of NanoIMB-Vc was reached 70.2％ at the level of 103 CFU/ml.In pure culture,the sensitivity of Nano-IMS plus Real-time PCR was reached at 5.4 × 102 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 choleraes test results were all positive,and the rest of the 101 strains of non-target bacteria test results were negative.No crossreaction 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.Conclusions The Nano-IMS plus Real-time PCR method of Vibrio choleraes established in this study has good specificity and sensitivity,which could be applied to the rapid detection of Vibrio choleraes.
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2014年第2期133-137,共5页
Chinese Journal of Preventive Medicine
基金
质检总局公益性行业科研专项（201110034）
国家质检总局科技计划项目（2009IK184）
关键词
弧菌
霍乱
聚合酶链反应
纳米医学
Vibrio, cholerae
Polymerase chain reaction
Nanomedicine
