Long Feng, Chen Yating, Shi Kaichuang, Yin Yanwen, Feng Shuping, Si Hongbin
Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China.
College of Animal Science and Technology, Guangxi University, Nanning 530005, China.
Animals (Basel). 2023 Feb 8;13(4):594. doi: 10.3390/ani13040594.
Porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (European genotype) and PRRSV type 2 (North American genotype) are prevalent all over the world. Nowadays, the North American genotype PRRSV (NA-PRRSV) has been widely circulating in China and has caused huge economic losses to the pig industry. In recent years, classical PRRSV (C-PRRSV), highly pathogenic PRRSV (HP-PRRSV), and NADC30-like PRRSV (NL-PRRSV) have been the most common circulating strains in China. In order to accurately differentiate the circulating strains of NA-PRRSV, three pairs of specific primers and corresponding probes were designed for the Nsp2 region of C-PRRSV, HP-PRRSV, and NL-PRRSV. After optimizing the annealing temperature, primer concentration, and probe concentration, a multiplex real-time quantitative RT-PCR (qRT-PCR) and a multiplex Crystal digital RT-PCR (cdRT-PCR) for the differential detection of C-PRRSV, HP-PRRSV, and NL-PRRSV were developed. The results showed that the two assays illustrated high sensitivity, with a limit of detection (LOD) of 3.20 × 10 copies/μL for the multiplex qRT-PCR and 3.20 × 10 copies/μL for the multiplex cdRT-PCR. Both assays specifically detected the targeted viruses, without cross-reaction with other swine viruses, and indicated excellent repeatability, with coefficients of variation (CVs) of less than 1.26% for the multiplex qRT-PCR and 2.68% for the multiplex cdRT-PCR. Then, a total of 320 clinical samples were used to evaluate the application of these assays, and the positive rates of C-PRRSV, HP-PRRSV, and NL-PRRSV by the multiplex qRT-PCR were 1.88%, 21.56%, and 9.69%, respectively, while the positive rates by the multiplex cdRT-PCR were 2.19%, 25.31%, and 11.56%, respectively. The high sensitivity, strong specificity, excellent repeatability, and reliability of these assays indicate that they could provide useful tools for the simultaneous and differential detection of the circulating strains of C-PRRSV, HP-PRRSV, and NL-PRRSV in the field.
1型猪繁殖与呼吸综合征病毒（PRRSV，欧洲基因型）和2型PRRSV（北美基因型）在全球广泛流行。如今，北美基因型PRRSV（NA-PRRSV）已在中国广泛传播，给养猪业造成了巨大经济损失。近年来，经典PRRSV（C-PRRSV）、高致病性PRRSV（HP-PRRSV）和NADC30样PRRSV（NL-PRRSV）一直是中国最常见的流行毒株。为了准确鉴别NA-PRRSV的流行毒株，针对C-PRRSV、HP-PRRSV和NL-PRRSV的Nsp2区域设计了三对特异性引物和相应探针。在优化退火温度、引物浓度和探针浓度后，建立了用于鉴别检测C-PRRSV、HP-PRRSV和NL-PRRSV的多重实时定量RT-PCR（qRT-PCR）和多重Crystal数字RT-PCR（cdRT-PCR）方法。结果表明，这两种方法均具有高灵敏度，多重qRT-PCR的检测限（LOD）为3.20×10拷贝/μL，多重cdRT-PCR的检测限为3.20×10拷贝/μL。两种方法均能特异性检测目标病毒，与其他猪病毒无交叉反应，且重复性良好，多重qRT-PCR的变异系数（CV）小于1.26%，多重cdRT-PCR的变异系数为2.68%。然后，共使用320份临床样本评估这些方法的应用，多重qRT-PCR检测C-PRRSV、HP-PRRSV和NL-PRRSV的阳性率分别为1.88%、21.56%和9.69%，而多重cdRT-PCR的阳性率分别为2.19%、25.31%和11.56%。这些方法的高灵敏度、强特异性、良好重复性和可靠性表明，它们可为现场同时鉴别检测C-PRRSV、HP-PRRSV和NL-PRRSV的流行毒株提供有用工具。
