瓜类果斑病菌荧光免疫层析试纸条的研制

Development of immunochromatographic test strip for detection of Acidovorax citrulli

为建立检测果斑病菌的新型免疫学检测技术,以异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)作为荧光探针,共价偶联抗瓜类果斑病菌野生型菌株SD01的鼠源单克隆抗体4F,将抗SD01单克隆抗体6D和羊抗鼠二抗喷涂于硝酸纤维素膜(NC膜)上分别作为检测线和质控线;以双抗体夹心反应模式制备检测果斑病菌的荧光免疫层析试纸条,并对试纸条的灵敏度、特异性及对实际样品的检测性能进行评估。结果表明:制备的荧光试纸条对纯培养果斑病菌SD01的检出限为3.1×10^(6)CFU∕mL;试纸条可检出8种果斑病菌(其中标准株1种,野生株7种),与6株近源的植物病原菌均无交叉反应,表明试纸条具有较高的特异性。在检测实际样品时,8种葫芦科植物的果斑病菌检出限为3.1×10^(6)CFU∕mL,表明汁液中的杂质对试纸条的检测灵敏度无影响,该试纸条适用于实际样品的检测。本研究制备的荧光试纸条操作简便、快速,结果肉眼可见,为果斑病菌的快速检测提供了一种新方法。

In order to establish a new immunological technology for the detection of Acidovorax citrulli,fluorescein isothiocyanate(FITC)was used as a fluorescent probe to covalently conjugated with mouse monoclonal antibody 4F against wild-type strain SD01 of A.citrulli.The anti-SD01 monoclonal antibody 6D and goat anti-mouse secondary antibody(IgG)were sprayed on the nitrocellulose membrane(NC membrane)as the detection line and the quality control line,respectively.The fluorescent immunochromatographic test strip for the detection of A.citrulli was prepared in the double antibodies sandwich reaction mode,and the sensitivity,specificity and detection performance of the test strip for real samples were evaluated.The results showed that the detection limit of SD01 was 3.1×10^(6) CFU/mL.Eight kinds of A.citrulli(including 1 standard strain and 7 wild strains)could be detected by the test strip,and there was no cross reaction between the test strip and 6 strains of plant pathogens,indicating that the test strip had high specificity.When testing actual samples,the detection limit of A.citrulli in 8 Cucurbitaceae plants was 3.1×10^(6) CFU/mL,indicating that the impurities in the juice have no effect on the detection sensitivity of the test strip,which was suitable for the detection of actual samples.The fluorescent test strip prepared in this study is simple and rapid,and the results are visible to the naked eye,which provides a new method for the rapid detection of A.citrulli.