猪鼻支原体血清抗体间接ELISA检测方法的建立

发布时间：2018-03-06 23:02

本文选题：间接ELISA　切入点：猪鼻支原体　出处：《畜牧兽医学报》2017年10期 　论文类型：期刊论文

【摘要】：猪鼻支原体是猪体内的一种常在菌,可引发猪的多发性浆膜炎、肺炎、关节炎、心包炎等炎症。由于猪鼻支原体与猪其他支原体有较高的交叉反应,目前并没有特异性的血清学诊断方法的报道。本试验以建立猪鼻支原体特异性ELISA检测方法为目的,将猪鼻支原体的表面可变脂蛋白Vlp的特定肽段作为包被抗原,优化该方法检测条件,最终确定:其抗原的最佳包被浓度为0.312 5μg·mL~(-1);一抗的最佳稀释度为1∶100,作用时间为30min;二抗的最佳稀释浓度为1∶20 000,最佳作用时间为30min;显色时间为10min;阴阳性的判定值为0.27。交叉性试验结果显示,Vlp抗原与猪其他支原体阳性血清及猪常见传染病阳性血清均无交叉反应。在重现性试验中,批内及批间的变异系数均低于10%,证明重现性良好。利用所建立的方法对已知阴性及阳性样本进行检测,结果显示特异性和灵敏度均良好。最后,用建立的方法对人工感染后28d的猪血清和247份临床样品进行检测,感染猪血清抗体呈阳性,建立的Vlp-ELISA方法、吐温20膜蛋白-ELISA方法对不同猪群临床样品检测的阳性率趋势相同。综上所述,建立的Vlp-ELISA方法特异性、稳定性良好,可以广泛用于临床猪鼻支原体血清抗体检测,为该病的检测和防控提供了重要工具。
[Abstract]:Mycoplasma suis is a common bacteria in pigs, which can cause polyserositis, pneumonia, arthritis, pericarditis and other inflammation in pigs. At present, there is no specific serological diagnostic method. In order to establish a specific ELISA detection method for mycoplasma suis, the specific peptide fragment of variable lipoprotein Vlp on the surface of Mycoplasma suis was used as the coating antigen. The detection conditions of this method are optimized. The final results showed that the best coating concentration of the antigen was 0.312 5 渭 g 路mL ~ (-1), the best dilution of the first antibody was 1: 100 and the action time was 30 min, the best dilution concentration of the second antibody was 1:20 000, the optimum time of action was 30 min, the color reaction time was 10 min, and the judging value of the yin-yang character was 0.27. The results of forkness test showed that there was no cross reaction between VLP antigen and other mycoplasma positive sera and the positive sera of common infectious diseases in pigs. The coefficient of variation within and between batches is lower than 10, which proves that the reproducibility is good. Using the established method to detect known negative and positive samples, the results show that the specificity and sensitivity are good. The established method was used to detect porcine serum and 247 clinical samples after 28 days of artificial infection. The antibody was positive in infected pig serum. The Vlp-ELISA method was established. The Tween-20 membrane protein Elisa method showed the same positive rate in clinical samples from different pig populations. In conclusion, the established Vlp-ELISA method was specific and stable, and could be widely used in clinical detection of mycoplasma mycoplasma antibody. It provides an important tool for the detection, prevention and control of the disease.
【作者单位】：江苏省农业科学院兽医研究所·农业部兽用生物制品工程技术重点实验室;省部共建国家重点实验室培育基地-江苏省食品质量安全重点实验室;
【基金】：国家重点研发计划(2016YFD0500702)
【分类号】：S858.28

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