摘要
根据猪圆环病毒2型(porcine circovirus 2,PCV2)、猪圆环病毒3型(porcine circovirus 3,PCV3)和猪圆环病毒4型(porcine circovirus 4,PCV4)保守区域基因序列合成特异性的引物和TaqMan探针,通过优化反应条件,建立了一种可同时快速高效鉴别检测PCV2、PCV3和PCV4的三重荧光PCR方法。结果显示,所建立的三重荧光PCR方法仅对PCV2、PCV3和PCV4出现阳性扩增,与猪伪狂犬病毒(PRV)、猪细小病毒(PPV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪支原体肺炎(M.hyo).猪流行性腹泻病毒(PEDV)和猪传染性胃肠炎病毒(TGEV)等猪常见病原无交叉反应;对PCV2.PCV3和PCV4的最低检出限分别为1.69×10^(1),1.38×10^(2),1.42×10^(2)拷贝;组内和组间试验变异系数均小于1.6%,重复性好。进一步应用所建立的PCV2、PCV3和PCV4三重荧光PCR方法对263份猪临床样品进行检测,结果检出168份PCV2阳性(阳性率为63.88%),35份PCV3阳性(阳性率为13.31%),21份PCV4阳性(阳性率为7.98%),其中PCV2/PCV3/PCV4共感染3份(阳性率为1.14%),检测结果与单重荧光PCR方法一致。本研究所建立的PCV2、PCV3和PCV4三重荧光PCR方法可以有效的实现对PCV2、PCV3和PCV4的同时快速检测。
The specific primers and TaqMan probes were designed based on the conserved gene regions of porcine circovirus 2(PCV2),porcine circovirus 3(PCV3) and porcine circovirus 4(PCV4),and a triple real-time PCR for simultaneous, rapid, efficient and differential detection of PCV2,PCV3 and PCV4 was established through optimizing the reaction conditions.The results showed that the developed triple real-time PCR assay only showed positive amplification for PCV2,PCV3 and PCV4,and no other common porcine pathogens such as PRV,PPV,PRRSV,CSFV,M.hyo,PEDV and TGEV were detected.The limit of detection was 1.69×10^(1) copies for PCV2,1.38×10^(2) copies for PCV3 and 1.42×10^(2) copies for PCV4.The assay had good reproducibility with intra-and inter-assay coefficients of variation at less than 1.6%.The developed triple real-time PCR assay for PCV2,PCV3 and PCV4 was further applied to detect 263 swine clinical samples.The detection results showed that 168of the samples were PCV2positive(63.88%),35of the samples were PCV3positive(13.31%),and 21of the samples were PCV4positive(7.98%).Among the positive samples,3samples were co-infected with PCV2,PCV3and PCV4(1.14%).All the detection results of triple real-time PCR assay were consistent with the single real-time PCR method.In summary,the PCV2,PCV3and PCV4triple real-time PCR assay developed in this study could effectively realize the simultaneous and rapid detection of PCV2,PCV3and PCV4in clinical samples.
作者
高艺祥
王金凤
张倩
孙晓霞
刘立兵
顾文源
马宏伟
马增军
韩庆安
王建昌
GAO Yixiang;WANG Jinfeng;ZHANG Qian;SUN Xiaoxia;LIU Libing;GU Wenyuan;MA Hongwei;MA Zengjun;HAN Qinan;WANG Jianchang(Hebei Province Key Laboralo-ry of Veterinary Medicine,College of Animal Science and Technology,Hebei Normal University of Science&Technology,Qinhuangdao,Hebei 066004,China;Hebei Provincial Center for Animal Disease Prevention and Control,Shijiazh ucang 050035,China;Technology Center of Shijiazhuang Customs.Shijiazhuang 050051,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2022年第9期1752-1757,1763,共7页
Chinese Journal of Veterinary Science
基金
河北省省级科技资助项目(19226612D)。
关键词
PCV2
PCV3
PCV4
三重荧光PCR
porcine circovirus 2
porcine circovirus 3
porcine circovirus 4
triple real-time PCR
