摘要
根据新型鸭呼肠孤病毒（novel duck reovirus,NDRV）S1基因序列设计1对特异性引物,建立了基于SYBR GreenⅠ的实时荧光定量RT-PCR检测方法。根据含目的基因的质粒拷贝数与定量反应Ct值的关系,绘制了标准曲线。敏感性试验显示,该方法最低可检出15个拷贝的病毒c DNA,其病毒最低检出量为0.5TCID50。该方法具有良好的特异性,对鸭坦布苏病毒（DTMUV）、A型鸭甲肝病毒（DHV-1）、C型鸭甲肝病毒（DHV-3）、番鸭呼肠孤病毒（MDRV）、减蛋综合征病毒（EDSV）、鸭新城疫病毒（NDV）、鸭瘟病毒（DPV）、鸭疫里默氏杆菌（RA）的检测结果均为阴性。该方法重复性好,组内变异系数为0.32%~0.91%,组间变异系数为1.03%~1.51%。利用该方法对人工感染雏鸭的粪便排毒情况进行检测,结果发现,攻毒后2~12 d是粪便排毒期,其中3~6 d是排毒高峰期。临床样品检测结果表明,该方法比常规RT-PCR具有更高的敏感性,而且从收到样品到得出检测结果只需4 h。
A pair of specific primers targeted to gene S1 of the novel duck reovirus （NDRV） was designed in this study, and a fluorescent quantitative RT-PCR （qRT-PCR） assay based on SYBR Green I fluorescent was also de- veloped for novel duck reovirus （NDRV） detection. The standard curve was plotted based on the linear relationship between the amount of plasmid DNA and the cycle threshold. The sensitivity test showed that the detection limit of qRT-PCR was about 15 copies for the cDNA of target gene, and the minimum detectable amount of the virus was 0. 5 TCID50. This method only detected NDRV, but not the duck tembusu virus （ DTMUV）, A-type duck hepatitis virus （DHV-1）, C-type duck hepatitis virus （DHV-3）, muscovy duck reovirus （MDRV）, egg drop syndrome virus （EDSV）, duck Newcastle disease virus （NDV）, duck plague virus （DPV）, and Riemerella anatipestifer （RA）.The method had good repeatability with intra-assay variation coefficients of 0. 32% -0.91% and inter-assay variation coefficients of 1.03% - 1.51%. The feces of artificially infected ducks were detected by the method, it was found that 2 - 12 d after attack was the detoxification period, of which 3 -6 d was the peak. It took only 4 hours from re- ceiving the clinical samples to obtaining the detection results by this method, which was more sensitive than conven- tional RT-PCR assay.
出处
《浙江农业学报》
CSCD
北大核心
2015年第8期1331-1336,共6页
Acta Agriculturae Zhejiangensis
基金
山东省海外高层次人才(泰山学者)引进计划
山东省现代农业产业技术体系家禽创新团队计划(SDAIT-13-011-01)
公益性行业(农业)科研专项(201003012)
作者简介
韩宏宇（1990-），女，内蒙古乌兰察布人，硕士研究生，主要从事禽病诊断技术的研究。E-mail：928203282@qq．com
于可响，E—mail：yukx1979@163．com
通讯作者，崔言顺，E-mail：yscui@sdau．edu．cn；
