猪圆环病毒3型TB GreenⅡ实时荧光定量PCR检测方法的建立和应用

Development and Application of TB GreenⅡReal⁃time PCR Method for Detection of Porcine Circovirus Type 3

为建立猪圆环病毒3型(Porcine circovirus 3,PCV3)快速、灵敏且特异的检测方法,针对PCV3全基因组的保守区域设计特异性引物,构建标准质粒,通过优化反应体系和反应程序,建立基于TB GreenⅡ的实时荧光定量PCR(qPCR)方法,并对其特异性、敏感性、重复性和可行性进行验证。结果显示,建立的TB GreenⅡqPCR检测方法特异性良好,在4.74×10^(2)~4.74×10^(7)拷贝/μL的标准质粒间具有良好的线性关系(R^(2)=0.999)。建立检测方法的最低检出限为10拷贝/μL,与猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV2)无交叉反应,组内变异系数为0.33%~0.62%,组间变异系数为0.40%~0.73%。对收集的132份临床样品进行检测,PCV3 TB GreenⅡqPCR方法检出率为9.10%(12/132),高于PCV3常规PCR方法的检出率(4.55%,6/132)。综上,建立的PCV3 TB GreenⅡqPCR检测方法具有良好的敏感性、特异性和重复性,可用于临床样品中PCV3感染的诊断、流行病学监测和实验室研究等。

In order to establish a rapid,sensitive and specific detection method for porcine circovirus 3(PCV3),specific primers were designed based on the conservative region of the whole genome of PCV3,and the standard plasmid was constructed.By optimizing the reaction system and procedure,a real⁃time quantitative PCR(qPCR)method based on TB GreenⅡwas established,and its specificity,sensitivity,repeatability and feasibility were verified.The results showed that the primer specificity of the established TB GreenⅡqPCR detection method was good,and there was a good linear relationship(R^(2)=0.999)when the plasmid standard samples were from 4.74×10^(2) to 4.74×10^(7) copies/μL.The detection limit of the method was 10 copies/μL,and there was no cross reaction with classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine parvovirus(PPV)and porcine circovirus type 2(PCV2).The intra⁃group variation coefficient was 0.33%—0.62%,and the inter⁃group variation coefficient was 0.40%—0.73%.A total of 132 clinical samples were tested,the detection rate of TB GreenⅡqPCR was 9.10%(12/132),which was higher than that of conventional PCR(4.55%,6/132).The established TB GreenⅡqPCR method has good sensitivity,specificity and repeatability,and can be used for the diagnosis,epidemiological monitoring and laboratory research of PCV3 infection in clinical samples.