| 鸭疫里默氏杆菌实时荧光定量PCR快速检测方法的建立与应用研究 |
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| 引用本文： | 谢永平,盘宝进,陈泽祥,许力干,杨威,韦梅良.鸭疫里默氏杆菌实时荧光定量PCR快速检测方法的建立与应用研究[J].中国畜牧兽医,2010,37(10):87-91. |
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| 作者姓名： | 谢永平 盘宝进 陈泽祥 许力干 杨威 韦梅良 |
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| 作者单位： | （1. 广西兽医研究所， 南宁 530001；2.广西出入境检验检疫局技术中心， 南宁 530022） |
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| 基金项目： | 广西水产畜牧局科技攻关项目 |
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| 摘 要： | 根据GenBank登录的鸭疫里默氏杆菌外膜蛋白(OmpA)基因保守区序列,设计引物和探针,建立了直接检测鸭疫里默氏杆菌的实时荧光定量PCR快速方法。10倍梯度稀释RA菌株制备DNA模板测定方法的灵敏度,结果可在8.0×103CFU/mL浓度下特异地检测出目的菌,最低能检测到RA的DNA模板为8.0拷贝/μL;对鸭源大肠杆菌、多杀性巴氏杆菌、沙门氏菌、变形杆菌、金黄色葡萄球菌进行检测,结果均为阴性,建立的实时荧光定时PCR方法特异性强。用RA菌株人工感染雏鸭,感染后定时采集咽喉拭子、泄殖腔拭子、心血、肝脏、肺脏、脑,分别用实时荧光定量PCR检测,对脏器进行病原菌分离。结果表明,1/10半数致死量接种组,感染后8 h从心血、肝脏、脑组织检测到RA核酸,16 h后全部试样呈阳性;24 h首先从肝脏分离到RA,心血、肝脏、肺脏、脑组织均分离出RA。心血和脏器RA实时荧光定量PCR阳性检出率为63.8%(51/80),RA分离率为28.8%(23/80)。10个半数致死量接种组,1 h即可从咽喉拭子、心血和肝检测到RA核酸,5 h全部样品为阳性;5 h从肺和脑分离到RA。RA人工感染鸭的心血、肝脏、肺脏、脑实时荧光定量PCR阳性检出率为86.3%(69/80),RA分离率为60%(48/80)。用加热裂解法提取样品RA DNA只需30 min,建立的实时荧光定量PCR检测RA只需2 h,适用于RA的快速诊断、流行病学调查、种鸭引进隔离检疫、活鸭及其产品国内市场和进出口检验检疫。
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| 关 键 词： | 鸭疫里默氏杆菌 实时荧光定量PCR 快速检测 敏感性 特异性 |
Establishment and Application of Real-time Fluorescence qPCR Assay for Quick Detection of Riemerella anatipestifer Infection in Ducks |
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| XIE Yong-ping,PAN Bao-jin,CHEN Ze-xiang,XU Li-gan,YANG Wei,WEI Mei-liang.Establishment and Application of Real-time Fluorescence qPCR Assay for Quick Detection of Riemerella anatipestifer Infection in Ducks[J].China Animal Husbandry & Veterinary Medicine,2010,37(10):87-91. |
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| Authors: | XIE Yong-ping PAN Bao-jin CHEN Ze-xiang XU Li-gan YANG Wei WEI Mei-liang |
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| Institution: | (1.Guangxi Veterinary Research Institute, Nanning 53001,China;2.Technology Centre of Guangxi Entry-Exit Inspection and Quarantine Bureau, Nanning 530022,China) |
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| Abstract: | The primers and probe were designed according to Riemerella anatipestifer (RA) outer membrane protein (ompA) gene conserved sequence from the GenBank and a real-time fluorescence qPCR quick detection method of RA was established. The detection limit of the method was 8.0 copies/μL of RA DNA. It had a high specificity, was negative to the duck-origin E. coli, Pasteurella multocida, Salmonella, Proteus, Staphylococcus aureus. Ducklings artifically infected with a RA strain, and throat swabs, cloacal swabs, blood, liver, lung and brain were collected ,detected by real-time qPCR and isolated 〖HT〗〖ST〗〖WT〗〖HJ〗
〖WT5”BZ〗pathogenic bacteria. The results of 1/10 half lethal dose inoculation group showed that RA DNA in liver and brain were detected 8 h after infection, all the samples were positive 16 h after, RA were first isolated from liver 24 h after, RA positive rate was 63.8% (51/80) by real-time qPCR while the RA isolation rate was 28.8% (23/80) only. The results of 10 half lethal dose inoculation group showed that RA DNA in throat swab, heart and liver were detected 1 h after infection, all the samples were positive at 5 h, RA were first isolated from the lungs and brain at 5 h, RA positive rate was 86.3% (69/80) by real-time qPCR while the RA isolation rate is 60% (48/80) only. It was only 30 min for extracting RA DNA from detection tissue by pyrolysis. The detection time of the established real-time qPCR method was only 2 h.The method could be used for rapid diagnosis , epidemiological investigation, quarantine of introduction ducks, import and export inspection and quarantine of live ducks and their products. |
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| Keywords: | Riemerella anatipestifer real-time qPCR rapid detection sensitivity specificity |
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