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SYBR GreenⅠ荧光RT-PCR方法快速鉴定新城疫病毒毒力

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摘要 为快速鉴定不同新城疫病毒株(NDV)毒力,我们以NDVF蛋白裂解位点附近的基因序列自行设计1对引物,对不同毒力NDV毒株进行荧光RT—PCR扩增,根据其扩增效率及扩增产物熔解温度(Tm)值大小进行NDV毒株毒力的鉴定,建立了能区分NDV强弱毒株的SYBR GreenⅠ荧光RT—PCR方法;同时将其与鸡胚平均致死时间(MDT)测定和F蛋白裂解位点序列分析结果进行比较。结果发现:建立的SYBR GreenⅠ荧光RT—PCR方法只需4h即呵判定结果,NDV强弱毒株扩增产物Tm值为(83±0.5)℃且有2个峰值,阴性对照Tm值为(81±0.5)℃且只有1个峰值;对NDV强毒株的扩增效率即Ct值在11-20之间,NDV弱毒株Ct值在25~30之间,阴性对照Ct值在30以上,Ct值在20~25之问为强弱毒株混合物。该方法检测结果与MDT测定和F蛋白裂解位点分析结果一致。应用SYBRGreenI模式荧光RT—PCR方法与普通RT—PCR方法和鸡胚接种鉴定方法分别对80份临床组织样品进行检测,强毒株的检出率均为53.75%(43/80),而弱毒株的检出率相应为31.25%(25/80)、27.50%(22/80)和35.00%(28/80),检出符合率达89.3%以上。这些结果表明建立的SYBR GreenⅠ荧光RT—PCR方法具有特异、准确、快速等特点,可用于临床病料样本NDV毒株的毒力鉴定。
出处 《畜牧与兽医》 北大核心 2008年第11期80-83,共4页 Animal Husbandry & Veterinary Medicine
基金 贵州大学博士基金项目[贵大人科合字(2006)] 贵州省自然科学基金项目[黔科合字(2005)2030]
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参考文献12

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二级参考文献9

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