畜牧与兽医2024，Vol.56Issue(4)：84-88,5.
猪繁殖与呼吸综合征病毒2型TaqMan MGB探针荧光定量PCR方法的建立
Establishment of a TaqMan MGB probe fluorescence quantitative PCR method for porcine reproductive and respiratory syndrome virus type 2
摘要
Abstract
In order to establish an accurate,specific,efficient and rapid fluorescence quantitative PCR method for the detection of porcine reproductive and respiratory syndrome virus type 2(PRRSV2),a pair of specific primers and a MGB probe were designed and synthesized according to the ORF6 gene of different lineage PRRSV2 strains registered in GenBank.Then,a TaqMan MGB fluorescence quantitative PCR method for the detection of PRRSV2 was established by optimizing the conditions and systems of reaction,primer and probe ratio,and by conducting sensitivity,specificity and reproducibility experiments.The results showed that the method was highly specific and did not amplify the nucleic acid of PRRSV1,classical swine fever virus(CSFV),porcine pseudorabies virus(PRV),porcine circovirus type 2(PCV2),porcine circovirus type 3(PCV3),porcine epidemic diarrhea virus(PEDV),porcine parvovirus(PPV)and other pathogens.The minimum detection limit of the standard plasmid was 1×101 copies/μL.The standard curve showed a good linear relation,and the correlation coeffi-cient was(R2=0.994),with good stability and reproducibility.The intra-and inter-assay coefficients of variation were less than 1.78%.30 clinical positive samples were also positive using this method with Ct values between 22 and 36.In summary,the PRRSV2 TaqMan MGB flu-orescence quantitative PCR method established in this study could be used for rapid detection of PRRSV2 in clinical practice and laboratory trial,providing technical support for PRRS control.关键词
猪繁殖与呼吸综合征病毒/ORF6 基因/TaqMan MGB/qPCRKey words
porcine reproductive and respiratory syndrome virus/ORF6 gene/TaqMan MGB/qPCR分类
农业科技引用本文复制引用
徐之恒,王聪,张振东,孙少辉,董友富,唐伟明,徐孝宙,邢军,李豪,孙刘妹,万进..猪繁殖与呼吸综合征病毒2型TaqMan MGB探针荧光定量PCR方法的建立[J].畜牧与兽医,2024,56(4):84-88,5.基金项目
江苏省基础研究计划(自然科学基金)青年基金项目(BK20201005) （自然科学基金）
江苏农林职业技术学院校企合作项目(2020kj021) （2020kj021）
