非洲猪瘟病毒、高致病性猪繁殖与呼吸综合征病毒二重实时荧光定量PCR检测方法的建立与初步应用

Development and Application of a Duplex FQ-PCR for Differential Detection of African Swine Fever Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

建立一种特异、敏感、快速的实时荧光定量PCR(FQ-PCR)方法,用于非洲猪瘟病毒(ASFV)和高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)的鉴别诊断。针对ASFV的B646L基因和HP-PRRSV的NSP2基因分别设计特异性引物/探针对,经优化反应体系、反应程序等反应条件,建立一种基于探针技术的FQ-PCR方法,验证方法的敏感性、特异性和重复性,对130份临床样品进行检测,并与OIE检测方法(ASFV)及国标方法(HP-PRRSV)进行比较分析。本研究成功建立的ASFV和HP-PRRSV二重FQ-PCR检测方法在10^(-1)~10^(5) copies/μL模板范围内有良好的线性关系;对ASFV和HP-PRRSV基因出现阳性扩增,但对猪日本乙型脑炎病毒(JEV)、猪瘟(CSFV)、猪细小病毒(PPV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)美洲经典株(VR2332株)、健康猪脾脏等7种病原核酸样品对照未出现扩增;批内、批间试验变异系数在0.53%~3.14%,重复性良好;对ASFV和HP-PRRSV的最低检测模板浓度均为10 copies/μL;利用建立的二重FQ-PCR方法对130份临床样品进行检测,检测结果与OIE检测方法(ASFV)及国标方法(HP-PRRSV)完全一致。本研究成功建立了鉴别ASFV和HPPRRSV二重FQ-PCR检测方法,为ASFV和HP-PRRSV的鉴别诊断提供了快速、敏感、特异且能满足临床检测需求的检测方法。

To develop a specific,sensitive and rapid real-time fluorescence quantitative PCR(FQ-PCR)method for the differential diagnosis of African swine fever virus(ASFV)and highly pathogenic porcine reproductive and respiratory syndrome virus(HPPRRSV),specific primer/probe pairs were designed for B646L of ASFV and NSP2 of HP-PRRSV for development of a FQ-PCR method based on probe technology.The FQ-PCR method was optimized for its reaction conditions to and verified for its sensitivity,specificity and repeatability.The results showed that the duplex FQ-PCR assay had a good linear relationship at a template range of 10^(-1)-10^(5)copies/μL and specifically amplified ASFV and HP-PRRSV genes.The duplex FQ-PCR assay did not amplify the nucleic acid samples of seven pathogens,such as Japanese encephalitis virus(JEV),Classical swine fever virus(CSFV),Porcine parvovirus(PPV),Porcine pseudorabies virus(PRV),Porcine circovirus type 2(PCV2),Porcine reproductive and respiratory syndrome virus(PRRSV)American classic strain VR2332 and healthy porcine spleens.The coefficients of variation of intra-batch and inter-batch tests ranged from 0.53%to 3.14%,indicating good repeatability.The minimum template concentrations for both ASFV and HP-PRRSV were 10 copies/μL.Subsequently,130 clinical samples were tested using this method and the results were compared with the OIE method for ASFV and the national standard method for HP-PRRSV.The results of the duplex FQ-PCR method were consistent with the ASFV OIE method and the HP-PRRSV national standard method.In summary,a du plex FQ-PCR assay was developed in this study to differentiate ASFV from HPPRRSV,providing a rapid,sensitive and specific detection method for the differential diagnosis of ASFV and HP-PRRSV.