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当前位置: 首页 > 文章 > 牛病毒性腹泻病毒SYBR Green I实时定量RT-PCR检测方法的建立及应用 新疆农业科学 2014,51 (2) 333-339
Position: Home > Articles > Establishment and Application of SYBR Green I RealI Real-Time Quantitative RT-PCR Assay Ffor Thethe Detection Of of Bovine Viral Diarrhea Virus Xinjiang Agricultural Sciences 2014,51 (2) 333-339

牛病毒性腹泻病毒SYBR Green I实时定量RT-PCR检测方法的建立及应用

作  者：
韩猛立;黄新;钟发刚;唐思静
单  位：
巴音郭楞职业技术学院;新疆农垦科学院畜牧兽医研究所/新疆生产建设兵团绵羊繁育生物技术重点实验室
关键词：
牛病毒性腹泻病毒;实时荧光定量RT-PCR;SYBR Green I
摘  要：
【目的】在建立一种快速定量的用于检测牛病毒性腹泻病毒(BVDV)的实时荧光定量RT-PCR检测方法。【方法】根据GenBank公布的BVDV 5'端非编码区(5'UTR)核苷酸序列,软件分析后设计特异性扩增引物,建立SYBR Green I实时荧光定量PCR方法。【结果】建立的方法只能检测到BVDV,而与猪瘟病毒、牛传染性鼻气管炎病毒、牛轮状病毒及牛冠状病毒没有交叉反应,具有高度的特异性;在102~108copies/μL模板范围内具有良好的线性关系,所制作的标准曲线相关系数为0.998,最低可检测下限可达到102copies/μL,具有灵敏度高的特点;不同情况下3次重复实验,变异系数均小于1.5%,具有重复性好的特点。【结果】成功建立了一种用于检测BVDV的实时荧光定量RT-PCR技术,并可用于临床样品的检测,适用于BVDV的快速诊断和流行病学调查。
译  名：
Establishment and Application of SYBR Green I RealI Real-Time Quantitative RT-PCR Assay Ffor Thethe Detection Of of Bovine Viral Diarrhea Virus
作  者：
HAN Meng-li;HUANG Xin;ZHONG Fa-gang;TANG Si-jing;Research Institute of Animal Husbandry and Veterinary Sciences,Xinjiang Academy of Agricultural and Reclamation Sciences/Key Laboratory of OvineSheep Breeding Biology Technology of Xinjiang Production and Construction Corps;Bayingolin Vocational and Technical College,Korla,BaYin GuoLeng Technology College;
关键词：
bovine viral diarrhea virus(BVDV);;real-time quantitative RT-PCR;;SYBR Green I
摘  要：
【Objective】The research was aimeds to develop a SYBR Green I real-time quantitative RT-PCR assay for rapid quantitative detection of bovine viral diarrhea virus( BVDV). 【Method】According to the nucleotide sequence of BVDV 5'-untranslated region( 5'-UTR) available in GenBank,the specific primers were designed to perform in the real-time fluorescent quantitative RT-PCR assay. 【Result】The results showed that the specificity of this assay was high without any cross-reaction with classical swine fever virus,bovine rhinotracheitis virus,bovine rotavirus and bovine coronavirus. A good linear correlation was demonstrated in the standard curve for the real-time PCR assay within the range of 102~ 108copies / μL,with a correlation coefficient of 0. 998. The detection limit of the real-time PCR assay was 102copies of plasmids,indicating a good sensitivity. And the intra-assay and the inter-assay coefficient of variation values were maintained at less than 1. 5%. 【Conclusion】The established SYBR Green I real-time quantitative RT-PCR can be used for rapid detection,diagnosis and epidemiological investigation for of BVDV.

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