人肺炎链球菌快速检测胶体金试纸的研制

Preparation of colloidal gold test paper for rapid detection of human pneumococcus

[目的]研制一种快速检测人肺炎链球菌(S. pneumoniae)的免疫胶体金层析试纸。[方法]对肺炎链球菌表面蛋白A(pneumococcal surface protein A,PspA)基因序列(Gen Bank:U89711)作生物信息学分析,选取抗原表位强、种内同源性高的片段克隆表达纯化重组蛋白rPspA,并免疫BALB/c小鼠制备单克隆抗体;以双抗夹心法制备胶体金免疫层析试纸,并对其特异性、灵敏度以及稳定性进行验证分析。[结果]制备的胶体金试纸对rPspA最低检测限达到0. 1pg/m L,可在15min内完成对肺炎链球菌的检测,与其他10种重要呼吸道病原菌无交叉反应;试纸条在25℃条件下保存6个月仍具有良好的重复性和稳定性。[结论]PspA蛋白具有高度的表面暴露性和较强的抗原性,可作为Sp的检测标志物,以其抗体为基础制备的胶体金免疫层析试纸,适用于Sp菌感染的临床快速检测。

[Objective] An immunocolloidal gold chromatographic test paper was established for rapid detection of pneumococcal in humans( S. pneumoniae).[Method] The PspA gene sequence( Gen Bank: U89711) was analyzed by bioinformatics,the gene fragment with the strongest epitope and high homology was cloned,the recombine protein r PspA was expressed and purified. The monoclonal antibody was prepared by immunizing BALB/c mice;The immunochromatographic test strips were prepared with the principle of sandwich combined with colloidal gold technology,and the specificity,sensitivity and stability were verified.[Result] The minimum detection limit of r PspA by the colloidal gold test paper reached 0. 1 pg/m L,which can complete the detection of Streptococcus pneumoniae within 15 min,and there is no cross-reaction with other 10 kinds of common respiratory pathogens;Test strips reserved at 25° C for 6 months still have good repeatability and stability. [Conclusion] PspA protein has high surface exposure and strong antigenicity,and can be used as a detection marker of Sp. The colloidal gold immunochromatographic test paper prepared based on its antibody has the characteristics of fast,simple and sensitive,and is suitable for rapid clinical detection of Sp infections.