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| 4 种牛呼吸道病毒多重TaqMan RT⁃qPCR检测方法的建立及初步应用 |
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哈蕊1, 常生荣1, 宋晓育2, 鲁强生1, 李萌1, 黄书艳1, 苏明媛1, 张小丽1, 康棣1
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1.甘肃农业大学 动物医学院，甘肃 兰州 730070;2.汝阳县农业农村局，河南 洛阳 471200
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| 摘要: |
| 【 目的】建立牛冠状病毒（BCoV）、牛呼吸道合胞体病毒（BRSV）、牛副流感病毒 3 型（BPIV-3）及牛腺病毒 3 型（BAdV-3）等4种可引起牛呼吸道疾病（BRD）病毒的多重TaqMan RT-qPCR检测方法，为BCoV、BRSV、BPIV-3和BAdV-3的检测提供技术支持。【 方法】针对BCoV、BRSV、BPIV-3的N基因和BAdV-3的Hexon基因保守片段设计特异性引物及探针，对引物和探针浓度、退火温度等条件进行优化，建立四重TaqMan RT-qPCR检测方法，并对该方法的特异性、敏感性及重复性进行评价。用建立的方法对248份临床样品进行检测，同时与常规RTPCR检测方法的结果进行比较。【 结果】建立的检测方法标准曲线线性关系良好，可特异性地检测BCoV、BRSV、BPIV-3和BAdV-3，检测敏感性均为1×103拷贝/μL；批内重复变异系数均小于3%，批间重复变异系数均小于5%。248份临床样品检测结果显示，四重TaqMan RT-qPCR检测BCoV、BRSV、BPIV-3及BAdV-3的阳性率分别为12.5%，14.9%，3.63%和5.65%，与常规RT-PCR检测结果符合率分别为97.18%，95.56%，97.58%和97.98%。 【结论】建立了BCoV、BRSV、BPIV-3和BAdV-3四重TaqMan RT-qPCR检测方法，该方法具有良好的敏感性、特异性及重复性，检测结果稳定可靠。 |
| 关键词: 牛冠状病毒 牛呼吸道合胞体病毒 牛腺病毒3型 牛副流感病毒3型 多重TaqMan实时荧光定量PCR |
| DOI：10. 13207/j. jnwafu. 2026. 05. 012 |
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| 基金项目:国家自然科学青年基金项目（3240190813）；甘肃省高等学校创新基金项目（2022A-049） |
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| Establishment and preliminary application of a quadruplex TaqMan RT⁃qPCR assay for detection of 4 bovine respiratory viruses |
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HA Rui1, CHANG Shengrong1, SONG Xiaoyu2, LU Qiangsheng1, LI Meng1, HUANG Shuyan1, SU Mingyuan1, ZHANG Xiaoli1, KANG Di1
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1.College of Veterinary Medicine，Gansu Agricultural University，Lanzhou，Gansu 730070，China;2.Ruyang County Agriculture and Rural Affairs Bureau， Luoyang，Henan 471200，China
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| Abstract: |
| 【 Objective】This research aims to establish a multiplex TaqMan RT-qPCR assay for simultaneous detection of four viruses that causes bovine respiratory disease（ BRD）：bovine coronavirus（ BCoV），bovine respiratory syncytial virus（ BRSV），bovine parainfluenza virus type 3（ BPIV-3），and bovine adenovirus type 3 （BAdV-3），providing technical support for the diagnosis of such viruses.【Method】Specific primers and probes were designed for conserved regions of the N gene of BCoV，BRSV，BPIV-3，and the Hexon gene of BAdV-3.Reaction conditions，including primer and probe concentrations and annealing temperature，were optimized to establish a quadruplex TaqMan RT-qPCR assay. The specificity， sensitivity， and repeatability of the assay were then evaluated. The established assay was applied to test 248 clinical samples，and the results were compared with those obtained by conventional RT-PCR.【Result】The standard curves of the established assay demonstrated good linearity，and the assay specifically detected BCoV，BRSV，BPIV-3，and BAdV-3 with a sensitivity of 1×103 copies/μL for each target. Intra-assay coefficients of variation（ CVs） were less than 3%，and inter-as-say CVs were less than 5%. The detection results of 248 clinical samples showed that the positive rates of the quadruplex TaqMan RT-qPCR assay for BCoV，BRSV，BPIV-3，and BAdV-3 were12.5%，14.9%，3.63%，and 5.65%，respectively. The oncordance rates of these results with conventional RT-PCR assays were 97.18%，95.56%，97.58%，and 97.98% respectively.【Conclusion】A quadruplex TaqMan RT-qPCR assay for detecting BCoV，BRSV，BPIV-3，and BAdV-3 was established，which exhibited good sensitivity，specificity，repeatability，and reliable performance. |
| Key words: bovine coronavirus bovine respiratory syncytial virus bovine adenovirus type 3 bovine parain⁃fluenza virus type 3 multiple TaqMan RT-qPCR |
