开发和实施四重实时 RT-qPCR 方法鉴定猪繁殖与呼吸综合征病毒株。
Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains.
机构信息
National Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan 430070, China.
出版信息
Viruses. 2023 Sep 18;15(9):1946. doi: 10.3390/v15091946.
BACKGROUND
Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), leading to abortion in sows and respiratory distress in breeding pigs. In China, PRRSV1 and PRRSV2 are the two circulating genotypes in swine herds, with distinct virulence. PRRSV2 further consists of classical (C-PRRSV2), highly pathogenic (HP-PRRSV2), and NADC30-Like (N-PRRSV2) subtypes. The diversity of PRRSV poses challenges for control and eradication, necessitating reliable detection assays for differentiating PRRSV genotypes.
METHODS
A new TaqMan-based RT-qPCR assay with four sets of primers and probes targeting conserved regions of the ORF7 and NSP2 genes of PRRSV was developed, optimized, and evaluated by us. Reaction conditions such as annealing temperature, primer concentration, and probe concentration were optimized for the assay. Specificity, sensitivity, repeatability, stability, limit of detection (LOD), concordance with the reference method were evaluated for the assay.
RESULTS
The assay could detect and type PRRSV1, C-PRRSV2, HP-PRRSV2, and N-PRRSV2 simultaneously with 97.33% specificity, 96.00% sensitivity, 12 copies/μL LOD, 97.00% concordance with reference assays. We applied the assay to 321 clinical samples from swine farms in China. The assay successfully detected and typed 230 PRRSV-positive samples, with 24.78% (57/230) of them further confirmed by ORF5 gene sequencing. The prevalence of PRRSV subtypes among the positive samples was as follows: C-PRRSV2 (15.22%), HP-PRRSV2 (23.48%), and N-PRRSV2 (61.30%). Two samples showed coinfection with different PRRSV subtypes.
CONCLUSION
The quadruple RT-qPCR assay is a powerful tool for detecting and typing the currently circulating PRRSV strains in Chinese swine populations. It can assist in the surveillance of PRRSV prevalence and the implementation of prevention and control strategies.
背景
猪繁殖与呼吸综合征病毒（PRRSV）导致猪繁殖与呼吸综合征（PRRS），导致母猪流产和繁殖猪呼吸困难。在中国，PRRSV1 和 PRRSV2 是猪群中循环的两种基因型，具有不同的毒力。PRRSV2 进一步分为经典（C-PRRSV2）、高致病性（HP-PRRSV2）和 NADC30 样（N-PRRSV2）亚型。PRRSV 的多样性对控制和根除造成了挑战，需要可靠的检测方法来区分 PRRSV 基因型。
方法
我们开发、优化并评估了一种新的基于 TaqMan 的 RT-qPCR 检测方法，该方法使用针对 PRRSV ORF7 和 NSP2 基因保守区的四组引物和探针。优化了反应条件，如退火温度、引物浓度和探针浓度。评估了该检测方法的特异性、敏感性、重复性、稳定性、检测限（LOD）和与参考方法的一致性。
结果
该检测方法能够同时检测和分型 PRRSV1、C-PRRSV2、HP-PRRSV2 和 N-PRRSV2，特异性为 97.33%，敏感性为 96.00%，LOD 为 12 拷贝/μL，与参考方法的一致性为 97.00%。我们应用该检测方法对来自中国猪场的 321 份临床样本进行了检测。该检测方法成功检测和分型了 230 份 PRRSV 阳性样本，其中 24.78%（57/230）通过 ORF5 基因测序进一步确认。阳性样本中 PRRSV 亚型的流行情况如下：C-PRRSV2（15.22%）、HP-PRRSV2（23.48%）和 N-PRRSV2（61.30%）。有两个样本显示不同 PRRSV 亚型的混合感染。
结论
四重 RT-qPCR 检测方法是一种强大的工具，可用于检测和分型中国猪群中当前流行的 PRRSV 毒株。它可以协助监测 PRRSV 的流行情况，并实施预防和控制策略。
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