College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, P.R. China.
College of Plant Health and Medicine, Qingdao Agricultural University, Qingdao, 266109, P.R. China.
Arch Virol. 2024 Nov 13;169(12):242. doi: 10.1007/s00705-024-06161-w.
In this study, a multiplex PCR method was developed for the detection of four diarrhea-associated viruses of canines, including canine bocavirus (CBoV), canine circovirus (CCV), torque teno canis virus (TTCV), and canine kobuvirus (CKV). Four pairs of compatible primers, one specific for each virus, were designed based on conserved sequences. After optimization of parameters such as primer concentration and annealing temperature in single and multiple amplifications, four specific fragments were amplified simultaneously with high sensitivity and specificity in one PCR reaction. The fragments amplified were 165 bp (CBoV), 345 bp (CCV), 506 bp (TTCV), and 666 bp (CKV) in length. The sensitivity of this one-step multiplex PCR is about 10 times lower than that of regular singleplex PCR. There was no cross-reaction with the canine pathogens canine parvovirus (CPV), canine distemper virus (CDV), or canine coronavirus (CCoV). Testing of canine fecal samples from China using the multiplex PCR assay revealed the presence of CBoV, CCV, TTCV, and CKV in 10.1%, 6.2%, 2.8%, and 1.7% of the samples, respectively. The results of multiplex PCR agreed with the singleplex PCR results with a coincidence rate of 100%. In addition, the complete genome sequences of the viruses in three CKV-positive samples were determined and found to be 95.7 - 96.6% identical to the reference strain US-PC0082 and genetically more distant from other animal kobuvirus. The multiplex PCR method established in this study is convenient, with high specificity and sensitivity, which will be helpful for the rapid differential diagnosis of CBoV, CCV, TTCV, and CKV infections.
在这项研究中，开发了一种用于检测犬四种腹泻相关病毒的多重 PCR 方法，包括犬博卡病毒（CBoV）、犬圆环病毒（CCV）、扭体犬诺如病毒（TTCV）和犬杯状病毒（CKV）。基于保守序列，设计了四对适配引物，每个病毒一对。在单重和多重扩增中优化了引物浓度和退火温度等参数后，在一个 PCR 反应中可同时高灵敏度和特异性地扩增四个特定片段。扩增的片段分别长 165 bp（CBoV）、345 bp（CCV）、506 bp（TTCV）和 666 bp（CKV）。一步多重 PCR 的灵敏度比常规单重 PCR 低约 10 倍。与犬病原体犬细小病毒（CPV）、犬瘟热病毒（CDV）或犬冠状病毒（CCoV）无交叉反应。使用多重 PCR 检测来自中国的犬粪便样本显示，CBoV、CCV、TTCV 和 CKV 分别在 10.1%、6.2%、2.8%和 1.7%的样本中存在。多重 PCR 的结果与单重 PCR 结果完全一致，符合率为 100%。此外，还确定了三个 CKV 阳性样本中病毒的完整基因组序列，发现与参考株 US-PC0082 的同源性为 95.7-96.6%，与其他动物杯状病毒的遗传距离较远。本研究建立的多重 PCR 方法方便快捷，具有高度特异性和灵敏度，将有助于 CBoV、CCV、TTCV 和 CKV 感染的快速鉴别诊断。
