猪圆环病毒3型LAMP检测方法的建立与应用

Establishment and Application of LAMP Method for Detection of Porcine Circovirus Type 3

为快速诊断和检测猪圆环病毒3型(PCV3),本研究根据其ORF2基因保守序列,设计特异性引物,并通过Bst DNA聚合酶、引物最佳浓度比例、Mg^(2+)、dNTPs和甜菜碱浓度及反应条件优化,建立了环介导等温核酸扩增(LAMP)检测方法。结果显示,59℃恒温扩增36min即可出现梯形条带,且产物亦可通过SYBR GreenⅠ染色进行判断。该方法最低检测限为1.0×10~1copies·μL^(-1),与猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病毒(PRV)、猪瘟病毒(CSFV)及猪圆环病毒1型和2型(PCV1、PCV2)等均无交叉反应。检测68份呼吸道病猪淋巴结样品,PCV3阳性率占30.9%,与PCR检测结果符合率为95.6%(65/68)。相对传统PCR方法而言,该方法敏感特异,简便快速,可用于PCV3检测和临床诊断。

In order to diagnose and detect porcine circovirus type 3（PCV3）,in this research,specific primers were designed according to the conserved sequence of ORF2 gene.By optimizing the concentration of Bst DNA polymerase,the ratio of primers,Mg^2＋,dNTPs,betaine and reaction condition,the loop-mediated isothermal nucleic acid amplification（LAMP）was established.The results manifested that ladder strips were observed after 36 min with a constant temperature,and the product could be judged by SYBRⅠstaining.The detection limit of this way was 1.0×10^1 copies·μL^-1 which had no cross reaction with porcine reproductive and respiratory syndrome virus（PRRSV）,porcine pseudorabies virus（PRV）,classical swine fever virus（CSFV）,porcine circovirus type 1（PCV1）and type 2（PCV2）.From 68 lymph nodes of diseased pigs in respiratory system,the positive rate of 30.9%tested by LAMP had a high coincidence rate of 95.6%（65/68）with that by PCR detection.Compared with traditional technology,this method is more sensitive,simpler and can be used for detection and clinical diagnosis of PCV3.