猪圆环病毒三重PCR检测方法的建立及其广西地区流行病学调查

Development of a triplex PCR assay for detection of porcine circovirus and epidemiological investigation of porcine circovirus in Guangxi Province from 2018 to 2020

针对当前我国流行的猪圆环病毒(porcine circovirus,PCV)1型(PCV1)、2型(PCV2)和3型(PCV3),设计3对特异性引物,通过对引物浓度、退火温度和循环次数的优化,建立了鉴别检测PCV1、PCV2和PCV3的三重PCR方法。结果显示,该方法具有良好的特异性、敏感性和重复性,只能检出PCV1、PCV2和PCV3,其他猪病毒均未能扩出;对PCV1、PCV2和PCV3质粒标准品的检测下限分别为3.63×10^(3),3.63×10^(1),3.63×10^(2)拷贝/μL;相同条件下的扩增获得均匀一致的结果。利用所建立的三重PCR方法检测2018—2020年采自广西各地的1308份猪组织病料,结果显示,PCV1、PCV2和PCV3的阳性率分别为2.14%,56.65%,5.58%,并存在混合感染现象;PCV1、PCV2阳性率逐年下降,PCV3阳性率稳定控制,不同地市之间阳性率有所差异。结果表明,本研究成功建立了一种快速、特异、灵敏、经济的三重PCR方法,可用于PCV1、PCV2和PCV3的鉴别检测和流行病学调查;当前广西猪群普遍感染PCV1、PCV2和PCV3,应加强防控。

To differential detection of porcine circovirus type 1(PCV1),PCV2and PCV3,three pairs of specific primers were designed for PCV1,PCV2and PCV3,and after optimizing the reaction conditions of primer concentration,annealing temperature and amplification cycle,a triplex PCR assay was developed for simultaneous and differential detection of PCV1,PCV2and PCV3.The triplex PCR showed high specificity,sensitivity and repeatability.It could specifically detect PCV1,PCV2and PCV3without cross-reaction with other porcine viruses.The detection limits of PCV1,PCV2and PCV3standard plasmids were 3.63×10^(3)copies/μL,3.63×10^(1) copies/μL and 3.63×10^(2)copies/μL,respectively.Uniform results were acquired under the same amplification conditions.A total of 1308clinical samples collected from Guangxi Province from 2018to 2020were detected by the triplex PCR.The results showed that the positive rates of PCV1,PCV2and PCV3 were 2.14%,56.65%and 5.58%,respectively,and there existed co-infections of these pathogens.Furthermore,the positive rates PCV1and PCV2were gradually decreased year by year,while the positive rate of PCV3was relatively stable,and the positive rates of these pathogens varied from city to city in Guangxi Province.The results indicated that a rapid,specific,sensitive and economical triplex PCR assay was successfully developed for differential detection and epidemiological investigation of PCV1,PCV2and PCV3;the infection of PCV1,PCV2and PCV3was common in pig herds in Guangxi Province,which needed to pay more attention to prevent and control these pathogens.