摘要
为建立同时检测猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)的四重荧光定量PCR(qPCR)检测方法,针对PRRSV的ORF2基因、CSFV的5′UTR基因、PRV的gB基因、PCV2的ORF2基因设计了引物及Taq Man探针,并进行反应体系优化,敏感性、特异性验证,建立了同时检测上述4种病原的四重实时荧光定量PCR。结果显示,所建立的四重荧光PCR扩增效率(E)、相关系数(R^(2))及曲线斜率均在正常范围内,检测PRRSV、CSFV、PRV、PCV2 E值、R^(2)、斜率分别为92.9%、0.998、-3.504,90.7%、0.998、-3.566,94.3%、0.996、-3.466,94.2%、0.997及-3.470。PRRSV、CSFV、PRV、PCV2重组质粒最低检出限分别达到10^(2)、10^(2)、10^(2)、10^(3) copies/μL;四重qPCR反应体系中的多条引物间不发生交叉反应,经评价该方法特异性良好;批内和批间重复性试验结果显示,变异系数(CV)均在1%以下,具有良好的重复性。分别使用该方法和相应的国标荧光定量检测法对采集的298份临床样品进行检测对比,两种方法检测PRRSV、CSFV、PRV、PCV2阳性符合率分别为96.08%、96.38%、100%、94.95%,均在94%以上。结果表明,建立的检测方法方便、灵敏、高效、特异性强,适用于猪呼吸道疾病病原学、流行病学研究以及临床病例的诊断,并为猪呼吸道疾病的预防和控制提供技术支撑。
In order to establish a quadruplex fluorescence quantitative PCR detection method for the simultaneous detection of porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),porcine pseudorabies virus(PRV),and porcine circovirus type 2(PCV2),primers and Taq Man probes were designed for the ORF 2 gene of PRRSV,the 5′UTR gene of CSFV,the gB gene of PRV,and the ORF 2 gene of PCV2,and the reaction system was optimized,and the sensitivity and specificity were verified.A quadruplex real-time PCR method for the simultaneous detection of the above four pathogens was established.The results showed that the amplification efficiency(E),correlation coefficient(R^(2))and curve slope of quadruplex fluorescence PCR were within the normal range,and the detection of PRRSV,CSFV,PRV,PCV2 E values,R^(2) and slopes were 92.9%,0.998,-3.504,90.7%,0.998,-3.566,94.3%,0.996,-3.466,94.2%,0.997,-3.470,respectively.The minimum detection limits of PRRSV,CSFV,PRV and PCV2 recombinant plasmids reached 10^(2),10^(2),10^(2),and 10^(3) copies/μL,respectively.There was no cross-reactivity between multiple primers in the quadruplex reaction system,and the specificity of the method was evaluated as good.Both intra-batch and inter-batch repeatability results showed a coefficient of variation(CV)of less than 1%with good repeatability.The method and the corresponding international standard fluorescence quantitative detection method were used to detect and compare the 298 clinical samples collected,and the positive compliance rates of PRRSV,CSFV,PRV and PCV2 were 96.08%,96.38%,100%and 94.95%,respectively,all above 94%.The results showed that the method established in this study is convenient,sensitive,efficient and specific,which is suitable for the etiology of porcine respiratory diseases,epidemiological studies and the diagnosis of clinical cases,and provides technical support for the prevention and control of porcine respiratory diseases.
作者
李欣
杨莉
刘光亮
王文秀
刘海隆
曹宗喜
张艳
LI Xin;YANG Li;LIU Guang-liang;WANG Wen-xiu;LIU Hai-long;CAO Zong-xi;ZHANG Yan(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi,Xinjiang,830052,China;Institute of Animal Science and Veterinary Medicine,Hainan Academy of Agricultural Sciences,Haikou,Hainan,571100,China;Key Laboratory of Tropical Animal Breeding and Disease Research,Haikou,Hainan,571100,China;Shandong Binzhou Animal Science and Veterinary Medicine Academy,Binzhou,Shandong,256600,China)
出处
《动物医学进展》
北大核心
2024年第6期7-14,共8页
Progress In Veterinary Medicine
基金
海南省属科研院所技术创新专项项目(KYYS-2021-29)
海南省热带动物繁育与疫病研究重点实验室开放课题项目(HAAS2022PT0102)
中央引导地方科技发展专项项目(ZY2022HN09)。
