A RT-PCR Method for Detecting Bovine Rotavirus
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摘要:
目的 建立牛轮状病毒（Bovine rotavirus，BRV）便捷检测方法，为福建省BRV的流行病学调查检测提供便捷技术支持。 方法 根据GenBank公布的BRV基因组序列，利用Oligo7.0软件设计并合成1对特异性引物，通过优化反应条件，建立了检测BRV的RT-PCR方法，同时开展了特异性、敏感性和重复性试验，并将建立的方法应用于22份临床样品的检测。 结果 建立的方法仅能够扩增出BRV的特异性片段，对其他畜禽常见病原体无特异性扩增；该方法的灵敏度为6.86×105 copies·μL−1。对22份临床样品进行检测，检测出12份阳性样品，阳性率为54.55%。 结论 本研究建立的BRV RT-PCR检测方法具有良好的特异性、敏感性和重复性，可应用于临床样本的检测，为福建省BRV的诊断和流行病学调查提供了技术支持。 Abstract:Objective A method for bovine rotavirus (BRV) detection was developed to determine the current epidemiological status in Fujian province. Method A pair of specific primers was designed based on the BRV genome from GenBank using Oligo7.0. Reaction conditions of the RT-PCR method were optimized for the detection. Specificity, sensitivity, and repeatability of the assay were verified on 22 clinical samples. Result The newly developed method amplified only the specific fragment of BRV, not any of other common pathogens of livestock and poultry. A sensitivity of 6.86×105 copies·μL−1 was achieved. And, on the 22 clinical samples, 12 were tested positive at a rate of 54.55%. Conclusion The established RT-PCR method for BRV detection displayed high specificity, sensitivity and repeatability, which could be applied for clinical diagnosis and epidemiological investigation. -
Key words:
- Bovine rotavirus /
- detection method /
- RT-PCR
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图 1 RT-PCR扩增
M：DL2000 Marker；1~5：退火温度：52 ℃、54 ℃、56 ℃、58 ℃、60 ℃。
Figure 1. RT-PCR amplification
M: DL2000 Marker; 1–5: annealing temperatures of 52, 54, 56, 58, and 60 ℃, respectively.
图 2 特异性试验结果
M：DL2000 Marker；1：BRV阳性对照；2：阴性对照；3~16分别为禽呼肠孤病毒、丝状支原体山羊亚种、山羊支原体山羊亚种、牛肺炎支原体、绵羊肺炎支原体、山羊化脓隐秘杆菌、鸡细小病毒、鸡圆圈病毒3型、鸭圆环病毒1型、鸭圆环病毒2型、无乳支原体、大肠杆菌、沙门氏菌、羊口疮病毒。
Figure 2. Specificity of methodology
M: DL2000 Marker; 1: BRV positive control; 2: negative control; 3–16: templates of ARV, Mmc, Mcc, M.bovis, Mo, CP, ChPV, GyV3, DuCV1, DuCV2, MA, EC, SE, and ORFV, respectively.
图 3 敏感性试验结果
M：DL2000 Marker；1：BRV阳性对照；2~10：101~109倍比稀释。
Figure 3. Sensitivity of methodology
M: DL2000 Marker; 1: BRV positive control; 2–10: 101–109 times dilutions.
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