[1]曹军平,王晓泉,程汉,等.基于NP基因的新城疫病毒Class I实时荧光定量RT-PCR方法的建立及验证[J].江苏农业学报,2015,(06):1389-1394.[doi:doi:10.3969/j.issn.1000-4440.2015.06.030]
　CAO Jun-ping,WANG Xiao-quan,CHENG Han,et al.Development and validation of a real-time fluorescent quantitative RT-PCR based on NP gene of Newcastle disease virus (NDV) Class I[J].,2015,(06):1389-1394.[doi:doi:10.3969/j.issn.1000-4440.2015.06.030]
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基于NP基因的新城疫病毒Class I实时荧光定量RT-PCR方法的建立及验证()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年06期
页码:
1389-1394
栏目:
畜牧兽医·水产养殖
出版日期:
2015-12-31

文章信息/Info

Title:
Development and validation of a real-time fluorescent quantitative RT-PCR based on NP gene of Newcastle disease virus (NDV) Class I
作者:
曹军平12; 王晓泉1; 程汉2; 刘晓文1; 刘秀梵1
(1.扬州大学兽医学院,江苏扬州225009；2.江苏农牧科技职业学院，江苏泰州225300)
Author(s):
CAO Jun-ping12; WANG Xiao-quan1; CHENG Han2; LIU Xiao-wen1; LIU Xiu-fan1
(1.College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;2.Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300, China)〖KH+2*2D〗
关键词:
新城疫病毒Class I; 核衣壳蛋白基因; 荧光定量RT-PCR
Keywords:
Newcastle disease virus class I; NP gene; fluorescent quantitative RT-PCR
分类号:
S858.31
DOI:
doi:10.3969/j.issn.1000-4440.2015.06.030
文献标志码:
A
摘要:
新城疫病毒是影响养禽业健康发展的主要病原之一。新城疫病毒只有1个血清型，但根据亲源关系可以划分为Class I和Class Ⅱ两大类。为快速定量检测Class I新城疫病毒，根据其NP基因的保守序列,设计合成1对引物和TaqMan 探针,以Class I新城疫病毒JS-18-05株NP基因阳性重组质粒为标准品模板，建立荧光定量PCR标准曲线,首次建立了一种敏感、特异、重复性好的快速检测新城疫病毒Class I核酸载量的TaqMan荧光定量RT-PCR方法。该方法在 102～108拷贝范围内具有良好的线性关系,可检测到初始模板中1 μl 10拷贝的病毒核酸,与传统的病毒分离方法具有相近的敏感性,且这两种方法对33株Class I、Class Ⅱ新城疫病毒分离株尿囊液样品检测结果符合率为100%。
Abstract:
Newcastle disease (ND) is one of the most serious poultry diseases. Phylogenetic analysis has revealed Newcastle disease virus (NDV) strains include two distinct classes (Class I and Class Ⅱ) within a single serotype. To develop a quick method to detect NDV Class I, a pair of primers and a TaqMan probe were designed and synthesized according to NP gene conservative sequence of Newcastle disease virus Class I. The positive recombinant plasmid cloned with NP gene of JS-18-05 strain isolated from duck was used as a positive quantitative template to establish a standard curve. Then a rea1-time fluorescent quantitative RT-PCR assay was established. The method had a good linear relationship within the concentration of 102 to 108 copies of NDV, with which 1 μl 10 copy of the virus nucleic acid could be detected in the initial template. Compared with traditional methods, the assay esstablished in this study show the same results in detecting 33 NDV isolates. 

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期：2015-03-25 基金项目：国家自然科学基金重点项目(30630048)；国家科技支撑计划项目(2006BAD06A03) 作者简介：曹军平(1970-),男,湖北阳新人,博士,教授,主要从事分子病毒学及生物技术研究。（E-mail）cccc62@163.com 通讯作者：刘秀梵,（E-mail） xfliu@yzu.edu.cn
更新日期/Last Update: 2015-12-31