三、呼肠孤病毒3型（Reo3） RT-PCR检测方法的建立 - 中国实验动物学报.DOC

呼肠孤病毒Ⅲ型（Reo3）RT-PCR检测方法的建立 王吉，卫礼，贺争鸣，岳秉飞 （中国食品药品检定研究院、国家实验动物微生物、遗传检测中心，北京 100050） 【摘要】目的 建立呼肠孤病毒Ⅲ型（Reo3）RT-PCR检测方法，应用于实验动物及人用动物源性材料Reo3的检测。方法 根据已发表的呼肠孤病毒Ⅲ型（Reo3）M1基因序列，设计合成引物。提取Reo3细胞毒RNA，以其为模板，进行PCR扩增。优化反应条件，进行特异性、敏感性、稳定性试验。结果 建立的Reo3 RT-PCR检测方法特异、敏感，稳定。以Reo3 RNA逆转录产物为模板，所能检测RNA最小模板浓度为0.42pg/μl，可检测病毒最小滴度为10-9.ml-1。结论 建立的呼肠孤病毒Ⅲ型（Reo3）RT-PCR检测方法可用于实验动物及人用动物源性材料Reo3的检测。 【关键词】呼肠孤病毒Ⅲ型；反转录聚合酶链式反应；实验动物；人用动物源性材料 【中图分类号】33 【文献标识码】A 【文章编号】 Development of A RT-PCR Method for Determination of Mammalian Orthoreovirus 3 Wang Ji, Wei Li, He Zhengming, Yue Bingfei (National Institutes for food and drug Control, National Center for quality of Laboratory Animal, Beijing 100050, China) [Abstract] Objective To develop a RT-PCR method for determination of Mammalian Orthoreovirus 3 (Reo3) in laboratory animal and the biological materials for human use from the animal. Methods Two pair of primers specific to the sequences of M1 gene were designed according to the published Mammalian Orthoreovirus 3.With which the RNA of Reo3 was extracted and reversely transcribed to cDNA as a template for PCR amplification.The developed RT-PCR method was optimized, verified for specificity and sensitivity. Results The developed RT-PCR method is good in ensitivity, specificity and stability, and its minimum detection limit using the recombinant plasmid containing Reo3 gene as atemplate was 0.42pg/μl,and the lowest detection virus titer is 10-9.ml-1. Conclusion The developed RT-PCR method can be used in detecting the Mammalian Orthoreovirus 3 (Reo3) in laboratory animaland the biological materials for human use from the animal. 　　[Key words] Mammalian Orthoreovirus 3; RT-PCR; laboratory animal;biological materials for human use 呼肠孤病毒3型（Reo3））μl扩增产物于1.5%琼脂糖凝胶（含0.5μg/ml EB）进行电泳鉴定。电泳缓冲液为1×TAE(0.04 mol/L Tris乙酸，0.001 mol/L EDTA，pH8.0)，110V电泳30min，在紫外成像仪下观察PCR产物条带在凝胶中的位置，以100bp DNA ladder Marker为参照物，判定结果。RT-PCR阳性扩增片段进行测序，通过与Genbank 中Reo3序列进行比对以确定PCR检测的准确性。 1.8特异性的试验 2组引物分别以呼肠孤病毒3型（Reo3）、小鼠肺炎（PVM