基于恒温隔绝式荧光PCR技术现场检测牛轮状病毒方法的建立及应用

发布时间：2018-12-12 22:16

【摘要】：轮状病毒(rotavirus,RV)是犊牛腹泻的重要病因,笔者旨在建立基于恒温隔绝PCR(insulated isothermal PCR,iiPCR)技术现场检测牛轮状病毒(bovine rotavirus,BRV)的方法。根据BRV和牦牛RVVP6基因序列,设计合成引物和探针,通过优化反应体系,配合商品化PetNAD核酸萃取试剂盒,建立了检测BRV和牦牛RV的iiPCR方法。结果显示该方法只能检出BRV和牦牛RV,不能检出牛冠状病毒、牛星状病毒、牛病毒性腹泻-黏膜病病毒、牛大肠杆菌、牛沙门菌、牛艾美尔球虫、牛瑞氏隐孢子虫等无关病原;检测下限为96.6copies·μL-1,重复性好;对30份牛腹泻样本和30份牦牛腹泻样本的检测结果表明,本方法对BRV的检出率是33.33%,对牦牛RV的检出率为73.33%,与文献报道的检测BRV和牦牛RV的方法的符合率分别为100%;对2016年四川阿坝藏羌自治州14个牧场的88份犊牦牛腹泻样本RV的检测结果显示RV核酸阳性检出率为98.86%。本研究建立的BRV iiPCR方法特异性好、灵敏度高、重复性好,既可用于BRV检测,又可用于牦牛RV的检测;从核酸提取到报告检测结果仅需1h,操作方便,可现场使用,为BRV的现场快速诊断提供有力的工具。
[Abstract]:Rotavirus (rotavirus,RV) is an important cause of calf diarrhea. The purpose of this paper is to establish a method for the field detection of bovine rotavirus (bovine rotavirus,BRV) based on isothermal isolation of PCR (insulated isothermal PCR,iiPCR. According to the sequence of BRV and yak RVVP6 gene, the primers and probes were designed and synthesized. By optimizing the reaction system and combining with the commercial PetNAD nucleic acid extraction kit, a iiPCR method for the detection of BRV and yak RV was established. The results showed that only BRV and yak RV, could not detect bovine coronavirus, bovine stellate virus, bovine viral diarrhea mucosal disease virus, bovine Escherichia coli, Salmonella bovis, Eimeria bovis, Cryptosporidium bovis and other unrelated pathogens. The detection limit was 96.6copies 渭 L -1 and the reproducibility was good. The detection results of 30 samples of bovine diarrhea and 30 samples of yak diarrhea showed that the detection rate of BRV and RV were 33.33 and 73.33, respectively. The coincident rate with the reported methods for the detection of BRV and yak RV was 100. RV analysis of 88 calf yak diarrhea samples from 14 farms in Aba Zang Qiang Autonomous Prefecture in Sichuan Province in 2016 showed that the positive rate of RV nucleic acid was 98.86%. The BRV iiPCR method established in this study has the advantages of good specificity, high sensitivity and good repeatability. It can be used for both BRV and yak RV detection. It takes only 1 hour to extract nucleic acid from nucleic acid to report the result of detection. It is easy to operate and can be used in the field. It provides a powerful tool for rapid diagnosis of BRV in the field.
【作者单位】：西南民族大学生命科学与技术学院;国家民委青藏高原动物疫病防控创新团队;金瑞鸿捷(厦门)生物科技有限公司;
【基金】：十三五国家重点研发计划(2016YFD0500907) 国家民委科研项目(14XNZ025)
【分类号】：S852.653

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