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鹅源新城疫病毒实时荧光定量PCR检测方法的建立
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尹仁福，刘新鑫，丁壮*，刘美，吴昊
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(吉林大学 畜牧兽医学院 预防兽医学系，吉林 长春130062)
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摘要：
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根据GenBank上鹅源新城疫病毒NA-1株M基因的序列，在保守区域设计并合成了1对引物，采用荧光嵌合法（SYBR GreenⅠ）建立了检测鹅源新城疫病毒的实时荧光定量PCR（real-time PCR）。以鹅源新城疫病毒NA-1株反转录产物cDNA为标准品，通过优化反应条件，建立了标准曲线，并进行了融解曲线分析。结果，标准曲线的Ct值检测范围为23～36，相关系数（r2）为0.992；无引物二聚体及非特异性产物，且Tm=(83±0)℃。结果表明，建立的检测鹅源新城疫病毒实时荧光定量PCR方法特异性强、灵敏性高，能为以后快速诊断鹅源新城疫病毒提供有效保障。 |
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关键词：
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鹅源新城疫病毒；实时荧光定量PCR；快速诊断 |
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中图分类号：S 852.659.5 文献标识码：
A 文章编号：1673-4696(2008)04-0320-03 |
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Development of a real-time PCR assay for detection of
Newcastle disease virus in goose |
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YIN Ren-fu,LIU Xin-xin,DING Zhuang,LIU Mei,WU Hao |
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(Department of Preventive Veterinary Medicine,College of Animal Science and
Veterinary Medicine,Jilin University,Changchun 130062,China) |
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Abstract: According to the M gene sequence of Newcastle disease virus(NDV) strain NA-1 from goose available in GenBank,a pair of primers was designed for establishing a SYBR Green Ⅰreal-time PCR for M gene of NDV.To establish the standard curve,the cDNA of the NDV strain NA-1 was served as a stan-dard.The analysis of melting curve was also carried out.The results indicated that the linear range of Ct values ranged from 23 to 36 with a good correlation coefficient(r2=0.992) and the melting curve also showed a single peak with an Tm value of (83±0)℃.It was concluded that the established real-time PCR assay was highly sensitive and specific,which could provide a tool for rapid diagnosis of NDV in clinical samples from geese. |
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Key words: Newcastle disease virus from goose;real-time PCR;rapid diagnosis |
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