摘要
为实现对牛呼吸道疫病综合征(bovinerespiratory diseasecomplex,BRDC)16种常见病原的同时检测,本研究成功建立了一种实时荧光PCR检测平台。该检测平台具有较强的特异性,16种BRDC病原之间无交叉反应;以含有特异性靶基因的标准品质粒DNA或基因组DNA为模板,不同病原的检出限在1.0×10^(2)~1.0×10^(0)copies/μL或1.0~0.1pg/μL之间,且具有良好的重复性。应用该检测平台对河北地区的290份临床样品进行检测。结果显示,16种BRDC病原均有检出,其中牛冠状病毒、睡眠嗜组织菌、牛疱疹病毒1型、溶血曼氏杆菌、多杀巴氏杆菌和牛鼻炎病毒B型检出率较高,分别为28.62%(83/290)、24.48%(71/290)、21.37%(62/290)、19.31%(56/290)、16.55%(48/290)和16.20%(47/290)。进一步分析表明,同一阳性样本中BRDC病原多重感染的现象普遍存在,以双重感染最为严重,检出率高达31.38%(91/290);三重感染和四重感染的检出率分别为15.52%(45/290)和5.17%(15/290);也存在一定比例的五重、六重和七重感染。本研究建立的BRDC常见病原实时荧光PCR检测平台能够在相同反应体系和反应条件下,实现对16种BRDC常见病原的同时检测。该检测平台具有操作方便、检测通量高等优点,可为进境种牛隔离场和国内养牛场中BRDC常见病原的快速检测和监测提供有力的技术支持。
The study successfully established a real-time PCR detection platform,aiming to achieve simultaneous detection of 16 common pathogens of bovine respiratory disease complex(BRDC).The platform demonstrated strong specificity with no cross-reactivity among the 16 BRDC pathogens.Using standard quality plasmid DNA containing specific target genes or bacterial genome DNA as templates,the detection limits of different pathogens ranged from 1.0×10^(2)—1.0×10^(0)copies/μL or 1.0—0.1 pg/μL,and showing good reproducibility.The platform was used to detect 290 clinical samples from Hebei.The results showed that all 16 BRDC pathogens were detected.BCo V,H.somni,Bo HV-1,M.Haemolytica,P.multocida and BRBV were detected higher rates among BRDC pathogens,with the detection rates of 28.62%(83/290),24.48%(71/290),21.37%(62/290),19.31%(56/290),16.55%(48/290)and 16.20%(47/290),respectively.Further analysis showed that co-infection of multiple BRDC pathogens were common in the same positive sample.Double infections were identified as the most severe,with a detection rate of up to31.38%(91/290).Triple infections and quadruple infections were detected at rates of 15.52%(45/290)and 5.17%(15/290),respectively.Additionally,a percentage of quintuple,sextuple and septuple infections were also detected.This study developed a real-time PCR detection platform for common BRDC pathogens.The platform is capable of simultaneously detecting 16 common BRDC pathogens within the same reaction system and under the same reaction conditions.The platform offers simple operation and high detection flux,providing robust technical support for the rapid detection and monitoring of common BRDC pathogens in both quarantine farms of inbound breeding cattle isolation farms and domestic cattle farms.
作者
刘立兵
王金凤
李睿文
张晓利
孙晓霞
王建昌
LIU Libing;WANG Jinfeng;LI Ruiwen;ZHANG Xiaoli;SUN Xiaoxia;WANG Jianchang(Technology Center of Shijiazhuang Customs,Shijiazhuang 050051,China;College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China;Tangshan Animal Quarantine Station,Tangshan 063199,China)
出处
《中国兽医科学》
北大核心
2025年第1期86-93,共8页
Chinese Veterinary Science
基金
海关总署科研项目(2023HK035)。
关键词
牛呼吸道疫病综合征
实时荧光PCR
检测平台
多重感染
bovine respiratory disease complex(BRDC)
real-time PCR
detection platform
multiple infection
