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

›› 2010, Vol. 37 ›› Issue (10): 87-91.

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

谢永平1，盘宝进2，陈泽祥1，许力干1，杨威1，韦梅良2

（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）

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2010-10-20 发布日期:2010-10-20
通讯作者: 盘宝进

Establishment and Application of Real-time Fluorescence qPCR Assay for Quick Detection of Riemerella anatipestifer Infection in Ducks

XIE Yong-ping1,PAN Bao-jin2, CHEN Ze-xiang1, XU Li-gan1 ,YANG Wei1 ,WEI Mei-liang2

(1.Guangxi Veterinary Research Institute, Nanning 53001,China;2.Technology Centre of Guangxi Entry-Exit Inspection and Quarantine Bureau, Nanning 530022,China)

Received:1900-01-01 Revised:1900-01-01 Online:2010-10-20 Published:2010-10-20
Contact: PAN Bao-jin

摘要/Abstract

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8.0×103 CFU/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。

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

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.

Key words: Riemerella anatipestifer; real-time qPCR; rapid detection; sensitivity; specificity

中图分类号:

S852.61

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]. , 2010, 37(10): 87-91.

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]. , 2010, 37(10): 87-91.

[1]	祝希辉, 李艳兰, 王志伟, 庞喆羽, 李玉保, 刘成, 司振书, 路建彪, 曹胜亮, 薛希娟. 1株鸭疫里默氏杆菌的全基因组测序及耐药性与遗传进化分析[J]. 中国畜牧兽医, 2023, 50(3): 1140-1149.
[2]	陈栋, 张言浩, 张阳, 李森, 周祖涛, 李自力. 3株鸭疫里氏杆菌的分离鉴定与药敏试验[J]. 中国畜牧兽医, 2023, 50(3): 1250-1258.
[3]	肖洋洋, 李芮芮, 马忠臣, 唐恬, 王娜, 陈创夫, 郑炜, 王勇, 王鹏雁. 新疆石河子地区牛支原体的分离鉴定及药物敏感性分析[J]. 中国畜牧兽医, 2023, 50(2): 754-763.
[4]	魏津, 王甲, 赵浩然, 刘博, 刘元杰, 姚文生, 刘燕, 马欣. 不同地区鸡毒支原体的分离鉴定及药物敏感性研究[J]. 中国畜牧兽医, 2023, 50(1): 368-376.
[5]	马光强, 刘丽娟, 陈国权, 吴良涛, 杨均, 刘军泽, 李潇蒙, 潘成文, 周碧君. 鸭疫里默氏杆菌贵州流行株蜂胶灭活疫苗制备[J]. 中国畜牧兽医, 2022, 49(8): 3163-3170.
[6]	麻宝艺, 盛陈艳, 刘宏莹, 马青霞, 汪婷婷, 李健明, 时坤, 杜锐, 冷雪. BVDV 1型和2型TaqMan双重实时荧光定量PCR检测方法的建立[J]. 中国畜牧兽医, 2022, 49(6): 2326-2335.
[7]	吕晓楠, 徐立蒲, 张文, 王娜, 曹欢, 王小亮, 王姝, 王静波. 感染鲤浮肿病毒镜鲤的组织病理变化及病毒分布规律研究[J]. 中国畜牧兽医, 2022, 49(3): 1077-1084.
[8]	陈强, 程悦宁, 冯秋菊, 郭利, 张淑琴, 谭斌, 易立, 赵权, 程世鹏, 孙娜. 水貂肺炎克雷伯菌的分离鉴定及耐药性分析[J]. 中国畜牧兽医, 2022, 49(2): 700-708.
[9]	李林林, 董嘉文, 张俊勤, 黄允真, 孙敏华. 鸭疫里默氏杆菌RIA_0940基因缺失株的构建及主要生物学特性测定[J]. 中国畜牧兽医, 2022, 49(12): 4564-4572.
[10]	崔宝山, 黄飞, 王杰, 李楠, 高庆华. 基于单细胞转录组测序技术筛选牛体内囊胚性别特异性基因[J]. 中国畜牧兽医, 2022, 49(12): 4573-4581.
[11]	张甜甜, 吕文亮, 林显华, 鹿瑶, 徐海燕, 谷巍, 郝木强, 盛永杰. 1株鸡源肠炎沙门氏菌的分离鉴定及致病性分析[J]. 中国畜牧兽医, 2022, 49(10): 4097-4105.
[12]	陶维昆, 刘波, 黄飞, 王杰, 高庆华. Xist lncRNA介导X染色体失活及应用研究进展[J]. 中国畜牧兽医, 2022, 49(1): 216-223.
[13]	张军芳, 孙建富, 孙斌, 唐琳, 王英, 王恩泽, 崔岩, 李强, 严昌国, 李香子. 延边牛PPARγ基因的克隆及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(8): 2695-2704.
[14]	苗曼宁, 郭益文, 胡德宝, 曾雨晗, 李新, 张林林, 丁向彬, 郭宏. MSTN基因编辑牛肌肉转录组测序及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(7): 2271-2281.
[15]	李恒, 字向东. 牦牛跨膜附睾蛋白1基因的克隆及其在卵丘卵母细胞复合体中的表达分析[J]. 中国畜牧兽医, 2021, 48(4): 1284-1293.