Ma Yan, Shi Kaichuang, Chen Zhenhai, Shi Yuwen, Zhou Qingan, Mo Shenglan, Wei Haina, Hu Liping, Mo Meilan
College of Animal Science and Technology, Guangxi University, Nanning 530004, China.
Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China.
Pathogens. 2024 Apr 19;13(4):341. doi: 10.3390/pathogens13040341.
Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and pseudorabies virus (PRV) are significant viruses causing respiratory diseases in pigs. Sick pigs exhibit similar clinical symptoms such as fever, cough, runny nose, and dyspnea, making it very difficult to accurately differentially diagnose these diseases on site. In this study, a quadruplex one-step reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PRCoV, PRRSV, SIV, and PRV was established. The assay showed strong specificity, high sensitivity, and good repeatability. It could detect only PRCoV, PRRSV, SIV, and PRV, without cross-reactions with TGEV, PEDV, PRoV, ASFV, FMDV, PCV2, PDCoV, and CSFV. The limits of detection (LODs) for PRCoV, PRRSV, SIV, and PRV were 129.594, 133.205, 139.791, and 136.600 copies/reaction, respectively. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 0.29% to 1.89%. The established quadruplex RT-qPCR was used to test 4909 clinical specimens, which were collected in Guangxi Province, China, from July 2022 to September 2023. PRCoV, PRRSV, SIV, and PRV showed positivity rates of 1.36%, 10.17%, 4.87%, and 0.84%, respectively. In addition, the previously reported RT-qPCR was also used to test these specimens, and the agreement between these methods was higher than 99.43%. The established quadruplex RT-qPCR can accurately detect these four porcine respiratory viruses simultaneously, providing an accurate and reliable detection technique for clinical diagnosis.
猪呼吸道冠状病毒（PRCoV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪流感病毒（SIV）和伪狂犬病病毒（PRV）是引起猪呼吸道疾病的重要病毒。患病猪表现出相似的临床症状，如发热、咳嗽、流鼻涕和呼吸困难，这使得在现场准确鉴别诊断这些疾病非常困难。在本研究中，建立了一种用于检测PRCoV、PRRSV、SIV和PRV的四重一步逆转录实时定量PCR（RT-qPCR）方法。该检测方法具有很强的特异性、高灵敏度和良好的重复性。它只能检测PRCoV、PRRSV、SIV和PRV，与猪传染性胃肠炎病毒（TGEV）、猪流行性腹泻病毒（PEDV）、猪圆环病毒（PRoV）、非洲猪瘟病毒（ASFV）、口蹄疫病毒（FMDV）、猪圆环病毒2型（PCV2）、猪德尔塔冠状病毒（PDCoV）和猪瘟病毒（CSFV）无交叉反应。PRCoV、PRRSV、SIV和PRV的检测限（LOD）分别为129.594、133.205、139.791和136.600拷贝/反应。批内和批间变异系数（CV）范围为0.29%至1.89%。所建立的四重RT-qPCR用于检测2022年7月至2023年9月在中国广西采集的4909份临床样本。PRCoV、PRRSV、SIV和PRV的阳性率分别为1.36%、10.17%、4.87%和0.84%。此外，还使用先前报道的RT-qPCR对这些样本进行检测，这些方法之间的一致性高于99.43%。所建立的四重RT-qPCR能够同时准确检测这四种猪呼吸道病毒，为临床诊断提供了准确可靠的检测技术。
