中国兽医科学2025，Vol.55Issue(12)：1626-1634,9.DOI:10.16656/j.issn.1673-4696.2025.0213
鸭疫里默氏杆菌间接ELISA检测方法的建立与应用
Establishment and application of indirect ELISA detection method for Riemerella anatipestifer
摘要
Abstract
The aimis to establish a rapid and accurate indirect enzyme-linked immunosorbent assay(ELISA)method for detecting antibodies against Riemerella anatipestifer(RA),to facilitate its appli-cation in field settings.This study optimized key ELISA parameters using checkerboard titration,including antigen coating conditions,antigen dilution ratio,serum dilution ratio,HRP-labeled secondary antibody incubation time,and HRP-labeled secondary antibody dilution ratio.The sensitivity and specificity of the method were systematically evaluated.The results showed that the optimal antigen coating concentration for the established method was 109 CFU/mL,the optimal antigen coating conditions were 37℃ for 2 h,the optimal dilution ratio of the primary antibody serum was 1∶1 600,the optimal blocking solution was 50 g/L skim milk,the optimal blocking condition was 37℃ for 1 h,the optimal incubation condition for the first antibody was 37℃ for 2 h,the optimal reaction time for the HRP-labeled secondary antibody was 37℃ for 2 h,the optimal dilution ratio of the HRP-labeled secondary antibody was 1∶5 000,and the optimal chromogenic time was 20 min.Sensitivity testing revealed that the indirect ELISA could detect RA-CQ3(serotype 1)positive serum at a dilution of 1∶12 800 and RA-NJ2(serotype 2)positive serum at 1∶25 600,maintaining clear threshold discrimination.Specificity tests confirmed no cross-reactivity with positive sera against O1 avian pathogenic Escherichia coli,duck enteritis virus,reovirus DRV-TH11,reovirus DRV-Gu8,fowl adenovirus sero-type 4,and duck hepatitis A virus type 3.The established indirect ELISA method exhibits high specificity and sensitivity,providing a reliable technical platform for epidemiological surveillance and clinical RA antibody detection in RA infections.关键词
鸭疫里默氏杆菌/间接ELISA/优化/方法建立Key words
Riemerella anatipestifer/indirect ELISA/optimize/method establishment分类
农业科技引用本文复制引用
王梦迪,钟宁远,冷楠楠,蒋蔚,余祖功,阿曼古丽·斯拉依,韩先干..鸭疫里默氏杆菌间接ELISA检测方法的建立与应用[J].中国兽医科学,2025,55(12):1626-1634,9.基金项目
上海市农业科技创新项目:重要人兽共患病人工智能预警防控平台的创制及应用(沪农科T2023106) （沪农科T2023106）
