猪鼻支原体血清抗体间接ELISA检测方法的建立

doi:10.11843/j.issn.0366-6964.2017.10.016

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (10): 1932-1938.doi: 10.11843/j.issn.0366-6964.2017.10.016

猪鼻支原体血清抗体间接ELISA检测方法的建立

王佳^1,2, 熊祺琰^1*, 华利忠¹, 刘蓓蓓¹, 甘源¹, 冯志新¹, 刘茂军¹, 武昱孜¹, 邵国青^1*

1. 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, 南京 210014;
2. 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, 南京 210014

收稿日期:2017-04-17 出版日期:2017-10-23 发布日期:2017-10-23
通讯作者: 熊祺琰,副研究员,E-mail:qiyanxiongnj@163.com;邵国青,研究员,E-mail:gqshaojaas@163.com
作者简介:王佳(1987-),男,江苏常州人,助理研究员,硕士,主要从事动物疫病诊断研究,E-mail:dirkwang@126.com,Tel:025-84390880
基金资助:
国家重点研发计划（2016YFD0500702）。

Establishment of an Indirect ELISA Detection Method for Mycoplasma hyorhinis

WANG Jia^1,2, XIONG Qi-yan^1*, HUA Li-zhong¹, LIU Bei-bei¹, GAN Yuan¹, FENG Zhi-xin¹, LIU Mao-jun¹, WU Yu-zi¹, SHAO Guo-qing^1*

1. Key Laboratory of Animal Diseases Diagnostic and Immunology of Ministry of Agriculture, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
2. Key Lab of Food Quality and Safety of Jiangsu Province—State Key Laboratory Breeding Base, Nanjing 210014, China

Received:2017-04-17 Online:2017-10-23 Published:2017-10-23

摘要/Abstract

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 5 μg·mL^-1；一抗的最佳稀释度为1∶100，作用时间为30 min；二抗的最佳稀释浓度为1∶20 000，最佳作用时间为30 min；显色时间为10 min；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28 d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 hyorhinis (M. hyorhinis) is a commensal inhabitant in swine. It can cause polyserositis, pneumonia, arthritis and pericarditis in pigs. Because of the high cross-reactivity among M. hyorhinis and other swine mycoplasmas, there is no specific serological diagnostic method reported. In this study, an indirect ELISA was established by using the variable lipoprotein (Vlp) as the coating antigen. By optimizing the reaction conditions, the optimal concentration of coating antigen was 0.312 5 μg·mL^-1 and the serum dilution was 1∶100, and serum reaction time was 30 min. The optimal dilution at 1∶20 000 for the goat anti-pig HRP-IgG was determined, and the reaction time was 30 min. The optimal substrate chromogenic time was 10 min. The cut-off value of the assay was 0.27. In the cross-reactivity experiment, the Vlp-ELISA showed no cross-reactivity with common swine pathogens or other swine mycoplasmas. The coefficients of variation of the inter-assay and intra-assay experiments were both below 10%. To investigate the specificity and sensitivity of this method, the positive and negative sera samples were detected by the Vlp-ELISA. After that, a total of 247 clinical samples and sera from the experimentally-infected pigs were tested by the Vlp-ELISA, and the results showed the same tendency with that of Tween 20 extracted membrane protein-ELISA method. In conclusion, the indirect ELISA established in this study was specificity and stability, which can be widely used in clinical detection.

中图分类号:

S854.43

王佳, 熊祺琰, 华利忠, 刘蓓蓓, 甘源, 冯志新, 刘茂军, 武昱孜, 邵国青. 猪鼻支原体血清抗体间接ELISA检测方法的建立[J]. 畜牧兽医学报, 2017, 48(10): 1932-1938.

WANG Jia, XIONG Qi-yan, HUA Li-zhong, LIU Bei-bei, GAN Yuan, FENG Zhi-xin, LIU Mao-jun, WU Yu-zi, SHAO Guo-qing. Establishment of an Indirect ELISA Detection Method for Mycoplasma hyorhinis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(10): 1932-1938.

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猪鼻支原体血清抗体间接ELISA检测方法的建立

Establishment of an Indirect ELISA Detection Method for Mycoplasma hyorhinis

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