Abstract:

Objective A small peptide-based sandwich ELISA method was established for the detection of polymyxin E. coli resistance. Method Based on the principle of polymyxin-resistance, and the sequence and spatial structure of lipopolysaccharide ( LPS ) receptor complex in the E. coli. A sandwich ELISIA assay was established, and a detection kit was assembled by coating the 96-well plate with 9 peptides, in which samples were then added to be tested. The carboxyl -terminal labeled 13 peptide and HPR-labeled streptavidin were used as the primary antibody ( working concentration 1 ∶ 1 000) and secondary antibody ( working concentration 1 ∶ 2 000- 10 000) , respectively. Except the laboratory-purified exponential phase polymyxin-resistance positive E. coli samples, field samples ( including soil, river water and manure from breeding farms) , egg yolk powder and experimental animal drinking water were also tested. Result Using the ELISA assay, laboratory-purified exponential bacteria, field samples, egg yolk powder and experimental animal drinking water all could be detected. And, the sensitivity, specificity, precision and repeatability of the kit all achieved the expected goals. Conclusion The sandwich ELISA assay improved the standardization of drug-resistant bacteria detection.

Key words: synthetic small peptides mimicking LPS receptors, polymyxin resistance, E. coli, ELISA

摘要： 目的 夹芯式 ELISA 法检测多黏菌素大肠杆菌耐药性。 方法 基于多黏菌素大肠杆菌耐药性产生的原理及大肠杆菌细胞壁中脂多糖 LPS 受体复合物序列及空间结构设计了 9 肽和 13 肽,将 9 肽包被于 96 孔板上,然后滴加待检样品,使用羧基端标记生物素的13肽作为一抗, HPR标记的链霉亲和素为二抗，建立了夹芯式ELISIA 法并组装了检测试剂盒。除了对实验室纯化的指数期大肠杆菌耐药性阳性样品外,还对土壤、河水、养殖场的粪样等野外( 大田) 样品、实验动物饲料原料( 蛋 黄 粉 ) 、实验室动物饮用水进行了检测。 结果 利用ELISA 检测法,对实验室纯化的指数菌,野外大田试验菌和实验动物饲料原料及饮用水等均能检出,试剂盒的敏感性、特异性、精密性、重复性均达到预期目标。结论 建立的夹芯式 ELISIA 有可能提升了耐药菌检测的标准化。

关键词: LPS 受体小肽, 多黏菌素耐药, 大肠杆菌, ELISA 法