快速检测黄瓜细菌性角斑病菌的间接ELISA法优化

Optimization of the Indirect-ELISA Method For Rapid Detection of Pseudomonas syringae pv. lachrymans

采用黄瓜细菌性角斑病菌作为包被抗原,对反应条件进行优化,建立较优黄瓜细菌性角斑病菌间接酶联免疫吸附分析(ELISA)检测方法。通过对抗原包被浓度和抗体稀释浓度、封闭液和封闭时间、抗体温育时间等的优化,确定ELISA法最低检测限。结果表明:抗原的较优包被浓度为106cfu·mL-1,抗体的较优工作浓度为1∶4000,以5%胎牛血清作为封闭液,在37℃下封闭1.5h,加入抗体温育1.5h是间接ELISA法的较优反应条件,方法的检测限为105cfu·mL-1。对田间采集的病叶进行检测表明,该方法可准确检测出黄瓜细菌性角斑病菌,可用于田间黄瓜细菌性角斑病的快速诊断。

Pseudomonas syringae pv. lachrymans was coated to the ELISA plate. Through a series of optimization tests, the indirect- ELISA detection method for P. syringae pv. lachrymans was established. The lowest detection limit for ELISA was confirmed after the optimization of reaction conditions such as the type of blocking solution, the blocking time, the incubation temperature of antibody, and the proper dilution of antibody and its antigen coating concentration. The results showed that the best reaction condition of the indirect-ELISA was 1：4000 of antibody proper dilution, 106 cfu·mL^-1 of antigen coating concentration, 5% fetal bovine serum,blocking 1.5h at 37℃, antibody incubating lh at 37℃. The detection limit of method was 105 cfu·mL^-1. The method was used to detect samples of cucumber disease leaves collected from field,which could detected P.syringae pv. lachrymans accurately, and can be used to make a fast diagnosis for bacterial angular leaf spot of cucumber in field.