鸡白痢沙门氏菌胶体金免疫层析快速检测试纸条的研制及初步应用

Development and primary application of colloidal gold immunochromatography test strip for rapid detection of Salmonella pullorum

为了建立一种适合基层现场的鸡白痢沙门氏菌(Salmonella pullorum)抗体检测方法,以S.pullorum的基因组为模板,利用PCR方法扩增inv A基因并进行原核表达后,用Ni-NTA纯化inv A融合蛋白,Western-blot和ELISA进行分析;采用30 nm的胶体金溶液标记山羊抗鸡Ig Y,并对金标抗体的质量浓度和p H值进行优化;在NC膜上分别包被inv A融合蛋白和鸡Ig Y作为检测线和质控线,评估其特异性、灵敏性和稳定性.用该方法对临床的195份血清进行检测,平板凝集试验验证其准确性.结果表明:构建的inv A重组蛋白质粒在大肠杆菌BL21中成功高效表达,获得的蛋白纯度较高,经Western-blot和ELISA方法证实具有良好的反应原性.该试纸条最佳胶体金标记的p H值为7.6,抗体质量浓度为1.3 mg/m L,方法的灵敏性与平板凝集试验的灵敏性一致,与其他病原菌无交叉反应.对195份临床样本进行检测,其结果与平板凝集试验的符合率为96.43%.研究建立的S.pullorum胶体金免疫层析检测试纸条具有方便、快捷、安全和灵敏度高等优势,可用于S.pullorum抗体的早期检测和流行病学筛查.

In order to establish a suitable primary site detection method for Salmonella pullorum antibody.The S.pullorum genome was used as template,that using PCR method to amplified invA gene and prokaryotic expression,purification of invA fusion protein by Ni-NTA,and analyzed by Western-blot and ELISA.Goat anti chicken IgY was labeled with colloidal gold solution of 30 nm,using goat anti chicken IgY as labeling antibody;On the NC film coated invA the fusion protein and chicken IgY,respectively,as a detection line and a quality control line.The concentration and pH value of gold labeled antibody were optimized,which to evaluate the specificity,sensitivity and stability.At last,195 serum samples were detected by this method,and the accuracy was verified by plate agglutination test.The results showed that the recombinant recombinant invA protein was successfully expressed in Escherichia coli BL21.The purity of the obtained protein was high and it has good reactivity confirmed by Western-blot and ELISA methods.Strip using the recombinant protein preparation,the optimal dilution of the colloidal gold labeled pH value and antibody concentration was 7.6 and 1.3 mg/mL,respectively.Furthermore,the sensitivity of this method was consistent with that of plate agglutination test,without cross reaction with other pathogenic bacteria.The 195 clinical samples were tested,comparing with the plate agglutination,which the rate of coincidence test was 96.43%.This study established S.pullorum colloidal gold immunochromatographic test strip was convenient,quick,safe and sensitive,which methods it could be potentially applied in the early detection and epidemiological investigation of S.pullorum antibody.