Jeon Gyu-Tae, Kim Hye-Ryung, Kim Jong-Min, Baek Ji-Su, Shin Yeun-Kyung, Kwon Oh-Kyu, Kang Hae-Eun, Cho Ho-Seong, Cheon Doo-Sung, Park Choi-Kyu
Animal Disease Intervention Center, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of Korea.
Foreign Animal Disease Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Republic of Korea.
Animals (Basel). 2023 Feb 9;13(4):602. doi: 10.3390/ani13040602.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been frequently reported in companion dogs and cats worldwide during the ongoing coronavirus disease. However, RT-qPCR methods developed for humans have been used for the diagnosis of SARS-CoV-2 infections in suspected companion dogs and cats owing to the lack of the companion animal-tailored methods. Therefore, we developed a multiplex RT-qPCR (mRT-qPCR) using newly designed primers and probes targeting and genes of all currently circulating SARS-CoV-2 variants as well as the canine or feline gene as an endogenous internal positive control (EIPC) for reliable diagnosis of SARS-CoV-2 infection from suspected dogs and cats. The developed mRT-qPCR assay specifically detected the target genes of SARS-CoV-2 but no other canine or feline pathogens. Furthermore, canine and feline EIPCs were stably amplified by mRT-qPCR in samples containing canine- or feline-origin cellular materials. This assay has high repeatability and reproducibility, with an optimal limit of detection (<10 RNA copies per reaction) and coefficients of variation (<1.0%). The detection rate of SARS-CoV-2 of the developed mRT-qPCR was 6.6% for canine and feline nasopharyngeal samples, which was consistent with that of a commercial mRT-qPCR kit for humans. Collectively, the newly developed mRT-qPCR with canine and feline EIPC can efficiently diagnose and evaluate the viral load in field specimens and will be a valuable tool for etiological diagnosis, epidemiological study, and controlling SARS-CoV-2 infections in canine and feline populations.
在当前的冠状病毒病流行期间，全球范围内伴侣犬和猫感染严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的情况屡有报道。然而，由于缺乏针对伴侣动物的检测方法，用于人类的逆转录定量聚合酶链反应（RT-qPCR）方法被用于疑似感染SARS-CoV-2的伴侣犬和猫的诊断。因此，我们开发了一种多重RT-qPCR（mRT-qPCR）方法，使用新设计的引物和探针，针对所有当前流行的SARS-CoV-2变体的 和 基因，以及犬或猫的 基因作为内源性内部阳性对照（EIPC），以可靠地诊断疑似犬和猫的SARS-CoV-2感染。所开发的mRT-qPCR检测方法能特异性检测SARS-CoV-2的靶基因，而不检测其他犬或猫的病原体。此外，在含有犬或猫源细胞材料的样本中，mRT-qPCR能稳定扩增犬和猫的EIPC。该检测方法具有高重复性和再现性，最佳检测限（每个反应<10个RNA拷贝）和变异系数（<1.0%）。所开发的mRT-qPCR对犬和猫鼻咽样本的SARS-CoV-2检测率为6.6%，与一种用于人类的商业mRT-qPCR试剂盒的检测率一致。总体而言，新开发的带有犬和猫EIPC的mRT-qPCR能够有效地诊断和评估野外样本中的病毒载量，将成为犬猫群体中SARS-CoV-2感染的病因诊断、流行病学研究和防控的宝贵工具。
