Gao Xiang, Liu Xinsheng, Zhang Yongguang, Wei Yanming, Wang Yonglu
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, 730070, China.
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Animal Virology of the Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China.
BMC Vet Res. 2020 May 7;16(1):130. doi: 10.1186/s12917-020-02341-3.
Porcine Deltacoronavirus (PDCoV) is a newly emerging Coronavirus that was first identified in 2012 in Hong Kong, China. Since then, PDCoV has subsequently been reported worldwide, causing a high number of neonatal piglet deaths and significant economic losses to the swine industry. Therefore, it is necessary to establish a highly sensitive and specific method for the rapid diagnosis of PDCoV.
In the present study, a highly sensitive and specific diagnostic method using recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) was developed for rapid and visual detection of PDCoV. The system can be performed under a broad range of temperature conditions from 10 to 37 °C, and the detection of PDCoV can be completed in 10 min at 37 °C. The sensitivity of this assay was 10 times higher than that of conventional PCR with a lower detection limit of 1 × 10 copies/µl of PDCoV. Meanwhile, the LFD-RPA assay specifically amplified PDCoV, while there was no cross-amplification with other swine-associated viruses, including Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine kobuvirus (PKoV), Foot and mouth disease virus (FMDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 2 (PCV2), Classical swine fever virus (CSFV) and Seneca valley virus (SVV). The repeatability of the test results indicated that this assay had good repeatability. In addition, 68 clinical samples (48 fecal swab specimens and 20 intestinal specimens) were further tested by LFD-RPA and RT-PCR assay. The positive rate of LFD-RPA clinical samples was 26.47% higher than that of conventional PCR (23.53%).
The LFD-RPA assay successfully detected PDCoV in less than 20 min in this study, providing a potentially valuable tool to improve molecular detection for PDCoV and to monitor the outbreak of PDCoV, especially in low-resource areas and laboratories.
猪德尔塔冠状病毒（PDCoV）是一种新出现的冠状病毒，于2012年在中国香港首次被发现。从那时起，PDCoV随后在全球范围内被报道，导致大量新生仔猪死亡，并给养猪业造成重大经济损失。因此，有必要建立一种高度灵敏且特异的方法用于快速诊断PDCoV。
在本研究中，开发了一种使用重组酶聚合酶扩增结合侧向流动试纸条（LFD-RPA）的高度灵敏且特异的诊断方法，用于快速且可视化地检测PDCoV。该系统可在10至37°C的广泛温度条件下进行，在37°C时10分钟即可完成PDCoV的检测。该检测方法的灵敏度比传统PCR高10倍，PDCoV的检测下限为1×10拷贝/微升。同时，LFD-RPA检测方法能特异性地扩增PDCoV，而与其他猪相关病毒无交叉扩增，这些病毒包括猪流行性腹泻病毒（PEDV）、传染性胃肠炎病毒（TGEV）、猪杯状病毒（PKoV）、口蹄疫病毒（FMDV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）、经典猪瘟病毒（CSFV）和塞内卡山谷病毒（SVV）。检测结果的重复性表明该检测方法具有良好的重复性。此外，通过LFD-RPA和RT-PCR检测方法对68份临床样本（48份粪便拭子标本和20份肠道标本）进行了进一步检测。LFD-RPA临床样本的阳性率比传统PCR高26.47%（传统PCR为23.53%）。
在本研究中，LFD-RPA检测方法在不到20分钟内成功检测到PDCoV，为改进PDCoV的分子检测以及监测PDCoV的爆发提供了一种潜在有价值的工具，尤其是在资源匮乏的地区和实验室。
