Li Biao, Shi Kaichuang, Shi Yuwen, Feng Shuping, Yin Yanwen, Lu Wenjun, Long Feng, Wei Zuzhang, Wei Yingyi
Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology, Guangxi University, Nanning 530005, China.
Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China.
Animals (Basel). 2025 Mar 31;15(7):1008. doi: 10.3390/ani15071008.
Porcine sapelovirus (PSV), porcine kobuvirus (PKV), porcine teschovirus (PTV), and porcine enterovirus G (EV-G) are all important viruses in the swine industry. These viruses play important roles in the establishment of similar clinical signs of diseases in pigs, including diarrhea, encephalitis, and reproductive and respiratory disorders. The early accurate detection of these viruses is crucial for dealing with these diseases. In order for the differential detection of these four viruses, specific primers and TaqMan probes were designed for the conserved regions in the 5' untranslated region (UTR) of these four viruses, and one-step quadruplex reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PSV, PKV, PTV, and EV-G was developed. The results showed that this assay had the advantages of high sensitivity, strong specificity, excellent repeatability, and simple operation. Probit regression analysis showed that the assay obtained low limits of detection (LODs) for PSV, PKV, PTV, and EV-G, with 146.02, 143.83, 141.92, and 139.79 copies/reaction, respectively. The assay showed a strong specificity of detecting only PSV, PKV, PTV, and EV-G, and had no cross-reactivity with other control viruses. The assay exhibited excellent repeatability of the intra-assay coefficient of variation (CV) of 0.28-1.58% and the inter-assay CV of 0.20-1.40%. Finally, the developed quadruplex RT-qPCR was used to detect 1823 fecal samples collected in Guangxi Province, China between January 2024 and December 2024. The results indicated that the positivity rates of PSV, PKV, PTV, and EV-G were 15.25% (278/1823), 21.72% (396/1823), 18.82% (343/1823), and 27.10% (494/1823), respectively, and there existed phenomena of mixed infections. Compared with the reference RT-qPCR/RT-PCR established for these four viruses, the coincidence rates for the detection results of PSV, PKV, PTV, and EV-G reached 99.51%, 99.40%, 99.51%, and 99.01%, respectively. In conclusions, the developed quadruplex RT-qPCR could simultaneously detect PSV, PKV, PTV, and EV-G, and provided an efficient and convenient detection method to monitor the epidemic status and variation of these viruses.
猪萨佩洛病毒（PSV）、猪杯状病毒（PKV）、猪特斯病毒（PTV）和猪肠道病毒G（EV-G）都是养猪业中的重要病毒。这些病毒在猪身上引发类似临床疾病症状方面发挥着重要作用，包括腹泻、脑炎以及生殖和呼吸障碍。对这些病毒进行早期准确检测对于应对这些疾病至关重要。为了对这四种病毒进行鉴别检测，针对这四种病毒5'非翻译区（UTR）的保守区域设计了特异性引物和TaqMan探针，并开发了用于检测PSV、PKV、PTV和EV-G的一步四重逆转录实时定量PCR（RT-qPCR）方法。结果表明，该检测方法具有灵敏度高、特异性强、重复性好、操作简便等优点。概率回归分析表明，该检测方法对PSV、PKV、PTV和EV-G的检测限较低，分别为146.02、143.83、141.92和139.79拷贝/反应。该检测方法仅对PSV、PKV、PTV和EV-G具有较强的特异性，与其他对照病毒无交叉反应。该检测方法的批内变异系数（CV）为0.28 - 1.58%，批间CV为0.20 - 1.40%，具有出色的重复性。最后，使用开发的四重RT-qPCR对2024年1月至2024年12月在中国广西采集的1823份粪便样本进行检测。结果表明，PSV、PKV、PTV和EV-G的阳性率分别为15.25%（278/1823）、21.72%（396/1823）、18.82%（343/1823）和27.10%（494/1823），存在混合感染现象。与针对这四种病毒建立的参考RT-qPCR/RT-PCR相比，PSV、PKV、PTV和EV-G检测结果的符合率分别达到99.51%、99.40%、99.51%和99.01%。总之，开发的四重RT-qPCR能够同时检测PSV、PKV、PTV和EV-G，为监测这些病毒的流行状况和变异提供了一种高效便捷的检测方法。
