Life Science and Engineering College, Northwest Minzu University, Lanzhou, 730030, China.
Neurology Department, Gansu Province People's Hospital, Lanzhou, 730030, China.
Pol J Vet Sci. 2020 Dec;23(4):481-489. doi: 10.24425/pjvs.2020.134696.
Bovine parvovirus (BPV), bovine coronavirus (BCoV) and bovine parainfluenza virus (BPIV) are common etiologies causing gastrointestinal and respiratory diseases in dairy herds. However, there are few reports on the synchronous detection of BPV, BCoV and BPIV. The present article aimed to develop a quick and accurate RT-PCR assay to synchronously detect BPV, BCoV and BPIV based on their specific probes. One pair universal primers, one pair specific primers and one specific probe was designed and synthesized. After the concentrations of primer and probe and annealing temperature were strictly optimized, the specificity, sensitivity and repeatability of the established triplex probe qRT-PCR were evaluated, respectively. The results showed the recombinant plasmids of pMD18-T-BPV, pMD18-T-BCoV and pMD18-T-BPIV were 554bp, 699bp and 704bp, respectively. The optimal annealing temperature was set at 45.0°C for triplex qRT-PCR. The triplex probe qRT-PCR can only synchronously detect BPV, BCoV and BPIV. Detection sensitivities were 2.0×102, 2.0×102 and 2.0×101 copies/μL for BPV, BCoV and BPIV, being 1000-fold greater than that in the conventional PCR. Detection of clinical samples demonstrated that triplex probe qRT-PCR had a higher sensitivity and specificity. The intra-assay and inter-assay coefficient of variation were lower than 2.0%. Clinical specimens verified that the triplex qRT-PCR had a higher sensitivity and specificity than universal PCR. In conclusion, this triplex probe qRT-PCR could detect only BPV, BCoV and BPIV. Minimum detection limits were 2.0×102 copies/μL for BPV and BCoV, and 2.0×101 copies/μL for BPIV. The sensitivity of this triplex probe qRT-PCR was 1000-fold greater than that in the conventional PCR. The newly qRT-PCR could be used to monitor or differentially diagnose virus infection.
牛细小病毒（BPV）、牛冠状病毒（BCoV）和牛副流感病毒（BPIV）是引起奶牛胃肠道和呼吸道疾病的常见病因。然而，关于同时检测 BPV、BCoV 和 BPIV 的报道很少。本研究旨在基于其特定探针，开发一种快速准确的 RT-PCR 检测方法，同时检测 BPV、BCoV 和 BPIV。设计并合成了一对通用引物、一对特异性引物和一条特异性探针。经过严格优化引物和探针浓度以及退火温度后，分别评估了建立的三重探针 qRT-PCR 的特异性、敏感性和重复性。结果显示，pMD18-T-BPV、pMD18-T-BCoV 和 pMD18-T-BPIV 的重组质粒分别为 554bp、699bp 和 704bp。三重 qRT-PCR 的最佳退火温度设定为 45.0°C。三重探针 qRT-PCR 只能同时检测 BPV、BCoV 和 BPIV。BPV、BCoV 和 BPIV 的检测灵敏度分别为 2.0×102、2.0×102 和 2.0×101 拷贝/μL，比常规 PCR 高 1000 倍。临床样本检测表明，三重探针 qRT-PCR 具有较高的灵敏度和特异性。组内和组间变异系数均低于 2.0%。临床标本验证，三重 qRT-PCR 比通用 PCR 具有更高的灵敏度和特异性。综上所述，该三重探针 qRT-PCR 只能检测 BPV、BCoV 和 BPIV。BPV 和 BCoV 的最低检测限分别为 2.0×102 拷贝/μL，BPIV 的最低检测限为 2.0×101 拷贝/μL。该三重探针 qRT-PCR 的灵敏度比常规 PCR 高 1000 倍。新的 qRT-PCR 可用于监测或鉴别病毒感染。
