猪圆环病毒2型荧光定量PCR检测试剂盒的研制及应用

摘要

In order to develop the diagnostic technique and diagnostic kit of porcine circovirus type 2 (PCV2),specific primers and a probe for the PCV2 ORF2 gene were specially designed,and the PCV2 was detected by a fluorescent quantitation PCR method.The tests showed that the cycle threshold Ct and the standard DNA template had an excellent linear relationship between 101-107 copies per microliter,and their correlation coefficient was 0.9999;The variation coefficient of Ct values was 0.59％-1.05％ in the intra-lot test and 1.9％-4.2％ in the inter-lot test,there being a good repeatability;The developed detection kit had no cross reaction with hog cholera virus (HCV),porcine respiratory and reproductive syndrome virus (PRRSV)and swine pseudorabies virus (PRV),and its detection and quantitation limits were 10 and 100 copy numbers respectively.The detection of 1 000 clinical samples by this method showed that the 710 samples were positive.The result indicated that this kit was high in sensitiveness,strong in specificity and good in stability,providing a simple and rapid method for PCV2 monitoring and epidemiological survey.%为了开发针对猪圆环病毒2型(PCV2)的诊断技术及其试剂盒,对PCV2的ORF2基因设计相应的特异性引物和探针,采用荧光定量PCR方法检测PCV2.试验表明:该方法循环阈值(Ct)与标准DNA模板在101-107拷贝/μL浓度范围内具有极好的线性关系,相关系数为0.9999;重复性好,批内试验中Ct值的变异系数在0.59％-1.05％,批间试验的Ct值变异系数为1.9％—4.2％;研制的检测试剂盒与猪瘟病毒(HCV)、猪呼吸与繁殖障碍综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)不发生交叉反应,检测极限和定量极限分别为10和100拷贝数.用此方法对1 000个临床样品进行检测,结果710个被检测为阳性.结果显示,所研制的PCV2诊断试剂盒灵敏度高,特异性强,稳定性好,为PCV2的检测和流行病学的调查提供了一种简便快速的诊断方法.