CAJ | 학술논문

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.
목적 채용납미면역자주(Nano-IMS)분리곽란호균,건립곽란호균실시형광PCR(Real-time PCR)검측방법.방법 채용곽란호균단극륭항체,제비곽란호균납미면역자주(Nano-IMB-Vc),특이성흡부곽란호균,결합Real-time PCR기술,건립곽란호균쾌속검측방법.선용15주전형균주,대Nano-IMB-Vc포획특이성진행험증;선취102주곽란호균화101주비목표균,대소건립적Real-time PCR방법진행특이성험증;채용순균적령민도검측화기질첨가실험대소건립방법적령민도진행검측,병여식품중곽란호균적검측방법(NMKL No.156)진행비교.결과 Nano-IMB-Vc재균체농도위103 CFU/ml수평,대곽란호균적포획솔최고가체70.2％.Nano-IMB-Vc분리결합Real-time PCR기술,재순배양、무수증균정황하,검측령민도체도5.4×102 CFU/ml;통과102주곽란호균화101주비목표균적측시,102주곽란호균검측결과균위양성,기여101주비목표균검측결과균위음성,무교차반응.채용Nano-IMB-Vc분리결합Real-time PCR기술,재식품기질첨가시험중,매25 g양품중첨가1CFU곽란호균,증균8h즉가검출.결론 Nano-IMB-Vc분리결합Real-time PCR기술,구유량호적특이성,교고적검측령민도,괄용우곽란호균적쾌속사선.
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.