柑橘溃疡病菌快速诊断胶体金速测卡的研制

A gold immunochromatographic assay for the detection of Xanthomonas citri pv. citri

将制备并提纯的抗Xcc单克隆抗体、多克隆抗体及重组抗体,采用间接ELISA方法对比其效价和特异性,筛选用于胶体金速测卡的最优抗体;采用柠檬酸三钠还原法制备胶体金颗粒,标记抗Xcc单抗Xcc-2D8,固定于金标垫上;利用双抗夹心原理,将单抗Xcc-2D6和羊抗鼠抗体分别固定于硝酸纤维素膜上,作为检测线(T线)和质控线(C线),经试验条件优化,组装胶体金速测卡密封干燥保存。对Xcc菌悬液及柑橘样本浸泡液进行检查。结果表明ELISA方法筛选出2株特异性强、稳定性好的抗体(Xcc-2D8效价为1:128 000倍、Xcc-2D6效价为1:256 000倍),应用于胶体金速测卡的研发;所研制速测卡对Xcc菌悬液的最低检测下限为1×103～1×104 cfu/mL,对多个地区的206份柑橘样本进行实际检测所得结果与qPCR方法所得结果具有较高一致性(kappa值=95.15%),混杂的柑橘组织液对检测结果无影响,检测时间控制在10min以内,检测常见细菌及植物病菌显示特异性良好,各批次速测卡无批间差异,速测卡保存方便稳定性良好。

The main aim of this study was to find a gold-immunochromatographic test-strip kit using a colloidal gold-labelled monoclonal antibody,which was developed for the detection of Xanthomonas citri pv.citri（Xcc）.Two mouse monoclonal antibodies against Xcc were obtained.One of them（Xcc-Mab2D8） was selected to conjugate with colloidal gold as the detector antibody that was spotted on a conjugate pad,and the other（Xcc-Mab2D6） was used as the capture antibody at the test line（T）.Goat anti-mouse IgG antibody was used as the capture antibody at the control line（C）.The entire test procedure only took about 10 min without any equipment.The detection limit of the test strip for Xcc concentration as low as 1×103-1×104 cfu/mL scored as positive.We tested 206 samples,and the results were compared with those obtained by qPCR in order to examine the reliability.The two methods showed significant correlation（kappa=95.15%）.