Cao Wei W, He Dong S, Chen Zhen J, Zuo Yu Z, Chen Xun, Chang Yan L, Zhang Zhi G, Ye Lei, Shi Lei
Institute of Food Safety and Nutrition, Jinan University, Guangzhou, China (Cao, Z.J. Chen, X. Chen, Chang, Shi, Ye); College of Veterinary Medicine, South China Agricultural University, Guangzhou, China (He); College of Animal Science and Technology, Agricultural University of Hebei, Baoding, China (Zuo); State Key Laboratory of Food Safety Technology for Meat Products and Synergetic Innovation Center of Food Safety and Nutrition, Xiamen Yinxiang Group, Xiamen, China (Zhang, Shi).
J Vet Diagn Invest. 2020 Jul;32(4):572-576. doi: 10.1177/1040638720924753. Epub 2020 Jun 18.
Porcine epidemic diarrhea, a disease caused by porcine epidemic diarrhea virus (PEDV), results in large economic losses to the global swine industry. To manage this disease effectively, it is essential to detect PEDV early and accurately. We developed a sensitive and accurate droplet digital PCR (ddPCR) assay to detect PEDV. The optimal primer-to-probe concentration and melting temperature were identified as 300:200 nM and 59.2°C, respectively. The specificity of the ddPCR assay was confirmed by negative test results for common swine pathogens. The detection limit for the ddPCR was 0.26 copies/μL, which is a 5.7-fold increase in sensitivity compared to that of real-time PCR (rtPCR). Both ddPCR and rtPCR assays exhibited good linearity, although ddPCR provided higher sensitivity for clinical detection compared to that of rtPCR. Our ddPCR methodology provides a promising tool for evaluating the PEDV viral load when used for clinical testing, particularly for detecting samples with low-copy viral loads.
猪流行性腹泻是一种由猪流行性腹泻病毒（PEDV）引起的疾病，给全球养猪业造成了巨大的经济损失。为了有效控制这种疾病，尽早准确地检测出PEDV至关重要。我们开发了一种灵敏且准确的液滴数字PCR（ddPCR）检测方法来检测PEDV。确定最佳引物与探针浓度和熔解温度分别为300:200 nM和59.2°C。通过对常见猪病原体的阴性检测结果证实了ddPCR检测方法的特异性。ddPCR的检测限为0.26拷贝/μL，与实时PCR（rtPCR）相比，灵敏度提高了5.7倍。尽管与rtPCR相比，ddPCR在临床检测中具有更高的灵敏度，但ddPCR和rtPCR检测方法均表现出良好的线性。我们的ddPCR方法为临床检测时评估PEDV病毒载量提供了一种有前景的工具，特别是用于检测低拷贝病毒载量的样本。
