猪繁殖与呼吸综合征病毒经典株和高致病株双重RT-PCR检测方法的建立  

Development of a duplex RT-PCR assay for the classical and highly pathogenic strains of porcine reproductive and respiratory syndrome virus

作　　者：赵刘洋 李涵乔 芮雪 段燕方 贺晓霜 周双海[1] ZHAO Liuyang;LI Hanqiao;RUI Xue;DUAN Yanfang;HE Xiaoshuang;ZHOU Shuanghai(College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China;Shunyi Center for Animal Epidemic Disease Prevention and Control,Beijing 101300,China)

机构地区：[1]北京农学院动物科学技术学院,北京102206 [2]顺义区动物疫病预防控制中心,北京101300

出　　处：《北京农学院学报》2022年第1期61-65,共5页Journal of Beijing University of Agriculture

基　　金：北京高等学校高水平人才交叉培养实培计划-科研训练计划深化项目(PXM2020_014207_000009)。

摘　　要：【目的】建立具有高敏感性能区分猪繁殖与呼吸综合征病毒经典株和高致病株的双重RT-PCR检测方法。【方法】首先从退火温度与引物浓度先对猪繁殖与呼吸综合征病毒经典株、高致病株的单项RT-PCR方法的条件分别进行筛选与优化,然后对其双重RT-PCR方法的条件进行筛选与优化,并进行特异性与敏感性检测,最后初步应用于临床样品检测。【结果】建立的猪繁殖与呼吸综合征病毒经典株和高致病株双重RT-PCR检测方法不能检出猪流行性腹泻病毒和猪瘟病毒等当前流行较广的其他猪源RNA病毒,对猪繁殖与呼吸综合征病毒经典株、高致病株的检测灵敏度分别是100、200 copies/μL。此方法对2020—2021年8个猪场的220份血清样品的检测结果显示,猪繁殖与呼吸综合征病毒经典株、高致病株的个体阳性率分别为7.73%和43.64%,与单项RT-PCR方法检测结果完全一致。【结论】建立了一种高敏感性的猪繁殖与呼吸综合征病毒经典株和高致病株双重RT-PCR检测方法。【Objective】 This study aimed to establish a duplex RT-PCR assay with high sensitivity for the classical and highly pathogenic(HP) strains of porcine reproductive and respiratory syndrome virus(PRRSV). 【Methods】 The annealing temperature and primer concentration conditions of single RT-PCR assay for the classical and HP strains of PRRSV were screened and optimized respectively, and then their duplex RT-PCR conditions were screened and optimized. Subsequently the specificity and sensitivity tests and clinical samples determination were conducted. 【Results】 The duplex RT-PCR assay for the classical and HP strains of PRRSV could not detect other current epidemic RNA viruses of porcine origin, such as porcine epidemic diarrhea virus and classical swine fever virus. The detection sensitivity for the classical and HP strains of PRRSV was 100, 200 copies/μL, respectively. The detection results of 220 sera samples from 8 pig farms during 2020-2021 with this duplex RT-PCR assay showed that the positive rates of the classical and HP strains of PRRSV were 7.73% and 43.64% respectively, which were consistent with the results of their single RT-PCR assay. 【Conclusion】 A duplex RT-PCR assay with high sensitivity for the classical and HP strains of PRRSV was established.

关 键 词：猪繁殖与呼吸综合征病毒 经典株 高致病株 双重RT-PCR 

分 类 号：S852[农业科学—基础兽医学]