基于 LPS 受体小肽的抗多黏菌素大肠杆菌的 ELISA 法的建立

doi:10. 3969 / j. issn. 1006-6179. 2022. 05. 005

实验动物科学 ›› 2022, Vol. 39 ›› Issue (5): 26-30.DOI: 10. 3969 / j. issn. 1006-6179. 2022. 05. 005

基于 LPS 受体小肽的抗多黏菌素大肠杆菌的 ELISA 法的建立

( 1. 杭州医学院临床医学院,杭州 310053) ( 2. 杭州医学院浙江省实验动物与安全性研究重点实验室,杭州 310013) ( 3. 浙江省农业科学院农产品质量安全与营养研究所,杭州 310021)

收稿日期:2022-08-09 出版日期:2022-10-28 发布日期:2022-11-15
通讯作者: 刘月环( 1974—) ,女,研究方向:微生物组基因编辑与动物模型质量控制. E-mail:215193122@ qq. com
作者简介:段悦庆( 2001—) ,男,研究方向:微生物检测与诊断. E-mail:1784344094@ qq. com
基金资助:
浙江省重点研发计划项目( 2020C02031)

A New Sandwich ELISA Method for Polymyxin-resistant Escherichia coli Based on Synthetic LPS Receptor Peptide

( 1. School of Clinical Medicine, Hangzhou Medical Collage, Hangzhou 310053, China) ( 2. Zhejiang Key Laboratory of Experimental Animal’ s & Nonclinical Laboratory Studies, Hangzhou Medical Collage, Hangzhou 310013, China) ( 3. Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China)

Received:2022-08-09 Online:2022-10-28 Published:2022-11-15

摘要/Abstract

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

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

中图分类号:

Q95-3

段悦庆, 柯舒文, 杨华, 唐标, 刘月环. 基于 LPS 受体小肽的抗多黏菌素大肠杆菌的 ELISA 法的建立[J]. 实验动物科学, 2022, 39(5): 26-30.

DUAN Yueqing, KE Shuwen, YANG Hua , TANG Biao , LIU Yuehuan. A New Sandwich ELISA Method for Polymyxin-resistant Escherichia coli Based on Synthetic LPS Receptor Peptide[J]. Laboratory Animal Science, 2022, 39(5): 26-30.

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基于 LPS 受体小肽的抗多黏菌素大肠杆菌的 ELISA 法的建立

A New Sandwich ELISA Method for Polymyxin-resistant Escherichia coli Based on Synthetic LPS Receptor Peptide

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