Shi Kaichuang, Shi Yandi, Shi Yuwen, Long Feng, Yin Yanwen, Pan Yi, Li Zongqiang, Feng Shuping
School of Basic Medical Sciences, Youjiang Medical University for Nationalities, Baise 533000, China.
College of Animal Science and Technology, Guangxi University, Nanning 530005, China.
Pathogens. 2024 Nov 29;13(12):1054. doi: 10.3390/pathogens13121054.
Canine coronavirus (CCoV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAV-2), and canine norovirus (CNV) are important pathogens for canine viral gastrointestinal and respiratory diseases. Especially, co-infections with these viruses exacerbate the damages of diseases. In this study, four pairs of primers and probes were designed to specifically amplify the conserved regions of the CCoV M gene, CRCoV N gene, CAV-2 hexon gene, and CNV RdRp gene. After optimizing different reaction conditions, a quadruplex RT-qPCR was established for the detection of CCoV, CRCoV, CAV-2, and CNV. The specificity, sensitivity, and repeatability of the established assay were evaluated. Then, the assay was used to test 1688 clinical samples from pet hospitals in Guangxi province of China during 2022-2024 to validate its clinical applicability. In addition, these samples were also assessed using the reported reference RT-qPCR assays, and the agreements between the developed and reference assays were determined. The results indicated that the quadruplex RT-qPCR could specifically test only CCoV, CRCoV, CAV-2, and CNV, without cross-reaction with other canine viruses. The assay had high sensitivity with limits of detection (LODs) of 1.0 × 10 copies/reaction for CCoV, CRCoV, CAV-2, and CNV. The repeatability was excellent, with intra-assay variability of 0.19-1.31% and inter-assay variability of 0.10-0.88%. The positivity rates of CCoV, CRCoV, CAV-2, and CNV using the developed assay were 8.59% (145/1688), 8.65% (146/1688), 2.84% (48/1688), and 1.30% (22/1688), respectively, while the positivity rates using the reference assays were 8.47% (143/1688), 8.53% (144/1688), 2.78% (47/1688), and 1.24% (21/1688), respectively, with agreements of more than 99.53% between two methods. In conclusion, a quadruplex RT-qPCR with high sensitivity, specificity, and repeatability was developed for rapid, and accurate detection of CCoV, CRCoV, CAV-2, and CNV.
犬冠状病毒（CCoV）、犬呼吸道冠状病毒（CRCoV）、犬腺病毒2型（CAV - 2）和犬诺如病毒（CNV）是引起犬类病毒性胃肠道和呼吸道疾病的重要病原体。特别是，这些病毒的共同感染会加剧疾病的损害。在本研究中，设计了四对引物和探针，用于特异性扩增CCoV M基因、CRCoV N基因、CAV - 2六邻体基因和CNV RdRp基因的保守区域。在优化不同反应条件后，建立了一种四重RT - qPCR方法用于检测CCoV、CRCoV、CAV - 2和CNV。评估了所建立方法的特异性、敏感性和重复性。然后，使用该方法对2022 - 2024年期间来自中国广西宠物医院的1688份临床样本进行检测，以验证其临床适用性。此外，还使用已报道的参考RT - qPCR方法对这些样本进行评估，并确定所开发方法与参考方法之间的一致性。结果表明，四重RT - qPCR只能特异性检测CCoV、CRCoV、CAV - 2和CNV，与其他犬类病毒无交叉反应。该方法具有高敏感性，CCoV、CRCoV、CAV - 2和CNV的检测限（LOD）为1.0×10拷贝/反应。重复性极佳，批内变异系数为0.19 - 1.31%，批间变异系数为0.10 - 0.88%。使用所开发方法检测CCoV、CRCoV、CAV - 2和CNV的阳性率分别为8.59%（145/1688）、8.65%（146/1688）、2.84%（48/1688）和1.30%（22/1688），而使用参考方法检测的阳性率分别为8.47%（143/1688）、8.53%（144/1688）、2.78%（47/1688）和1.24%（21/1688），两种方法之间的一致性超过99.53%。总之，开发了一种具有高敏感性、特异性和重复性的四重RT - qPCR方法，用于快速、准确地检测CCoV、CRCoV、CAV - 2和CNV。
