检测PRRSV及鉴别HP-PRRSV毒株的二重PCR与二重TaqMan荧光定量PCR方法
Discrimination of porcine reproductive and respiratory syndrome virus(PRRSV)and highly pathogenic PRRSV strain with a duplex PCR and a duplex TaqMan real-time PCR
陈旭 1汤德元 1曾智勇 1王彬 1袁盛林 1廖正波 1何松 1周飘 1毛茵茗1
作者信息
- 1. 贵州大学动物科学学院,贵州贵阳 550025
- 折叠
摘要
为了实现猪繁殖与呼吸综合征病毒(PRRSV)早期检测及快速鉴别高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)变异株,利用HP-PRRSV Nsp2蛋白第481位和532～560位氨基酸缺失的特性设计2对引物,优化反应条件建立二重PCR检测方法,同时针对PRRSV M基因和HP-PRRSV Nsp2基因设计特异性引物和探针,优化反应条件建立二重TaqMan荧光定量PCR方法,并评估这2种方法的敏感性、特异性、重复性.敏感性试验结果显示:二重PCR方法的检测下限分别为0.058 ng/μL(PRRSV)和4.7 ng/μL(HP-PRRSV);二重 TaqMan 荧光 PCR 方法的检测下限分别为 10 copies/μL(PRRSV)和 100 copies/μL(HP-PRRSV).这 2种方法与其他猪病病原均不发生非特异性反应,重复性试验结果良好.经131份临床样品检测,可得到PRRSV的阳性率约12.2％(16/131)、HP-PRRSV的阳性率约17.6％(23/131)、二者的混合感染率约8.4％(11/131).证实,本研究成功建立了二重PCR和二重TaqMan荧光定量PCR检测方法,可为临床上猪繁殖与呼吸综合征的防控提供技术支持.
Abstract
In order to achieve early detection of porcine reproductive and respiratory syndrome virus(PRRSV)and rapid identification of highly pathogenic porcine reproductive and respiratory syn-drome virus(HP-PRRSV)variants,two pairs of specific primers were designed based on characteristics of amino acid deletion at positions 481 and 532-560 of HP-PRRSV Nsp2 protein,a duplex PCR detection method was established by optimizing the reaction conditions.The specific primers and probes were de-signed based on PRRSV M gene and HP-PRRSV Nsp2 gene,a duplex TaqMan real-time PCR method was estab-lished by optimizing the reaction conditions.The sensitivity,specificity,and reproducibility of both methods were evaluated.The sensitivity test results showed that the lowest detection limits of the duplex PCR method were 0.058 ng/μL(HP-PRRSV)and 4.7 ng/μL(HP-PRRSV);the minimum detection limits of the duplex TaqMan real-time PCR method were 10 copies/μL(PRRSV)and 100 copies/μL(HP-PRRSV),and there was no cross-reactivity between these two detection methods and other swine pathogens,and the repeatability test results were good.A total of 131 clinical samples detection showed positive rate of PRRSV was 12.2％(16/131),the positive rate of HP-PRRSV was 17.6％(23/131),and mixed infection rate was 8.4％(11/131).It was confirmed that the duplex PCR and duplex TaqMan real-time PCR detection methods were successfully established,which could provide technical support for the prevention and control of porcine reproductive and respiratory syndrome in clinical practice.
关键词
猪繁殖与呼吸综合征病毒/高致病性猪繁殖与呼吸综合征病毒/二重PCR/二重TaqMan荧光定量PCR/鉴别检测Key words
PRRSV/HP-PRRSV/duplex PCR/duplex TaqMan real-time PCR/discriminated detection引用本文复制引用
基金项目
贵州省科技支撑计划(黔科合支撑[2021]一般162)
出版年
2024
国家科技期刊平台
NSTL
万方数据