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
详细信息 查看官网全文
- 英文论文题名：Validation and Development of a Real-Time Fluorescent Quantitative RT-PCR Based on NP Gene of Newcastle Disease Virus(NDV) to Detect NDV class Ⅰ
- 论文作者：曹军平 ; 王晓泉 ; 朱爱萍 ; 刘晓文 ; 刘秀梵
- 英文论文作者：CAO Jun-ping ; WANG Xiao-quan ; CHENG Han ; LIU Xiao-wen ; LIU Xiu-fan ; College of Veterinary Medicine ; Yangzhou University ; Jiangsu Agri-animal Husbandry Vocational College
- 年：2016
- 作者机构：扬州大学兽医学院;江苏农牧科技职业学院;
- 论文关键词：新城疫病毒class I、classⅡ ; 荧光定量RT-PCR ; 探针
- 英文论文关键词：Newcastle disease virus class Ⅰ and class Ⅱ ; fluorescent quantitative RT-PCR ; Taq Man probe
- 会议召开时间：2016-07-20
- 会议录名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会论文集
- 语种：中文
- 分类号：S852.65
- 学会代码：SYGW
- 会议名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会
- 会议地点：中国内蒙古通辽
- 主办单位：中国畜牧兽医学会兽医公共卫生学分会
- 学会名称：中国畜牧兽医学会兽医公共卫生学分会
- 页数：9
- 文件大小：558k
- 原文格式：D
- 会议级别：全国
摘要
新城疫病毒是影响养禽业健康发展的主要病原之一。新城疫病毒只有一个血清型,但根据亲源关系可以划分为class I和classⅡ两大类。classⅡ所有自然分离株均为无毒株或弱毒株,但有经人工传代返强的报道,由于其分布广泛,数量庞大,对养禽业有巨大的潜在威胁。为快速定量检测class I新城疫病毒,本研究根据其NP基因的保守序列,设计合成一对引物和Taq Man探针,以本室构建并保存的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,结合ABI公司的7300型荧光定量PCR仪,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Taq Man荧光定量RTPCR方法。该方法在106~101拷贝范围内具有良好的线性关系,可检测到初始模板中1拷贝/μL的病毒核酸,与传统的病毒分离方法具有相近的敏感性,二者对33株class I、classⅡ新城疫病毒分离株尿囊液样品检测结果符合率为100.0%。该法的建立为区分及定量分析禽新城疫病毒class I、classⅡ奠定了基础。
Newcastle disease(ND) is one of the most serious poultry diseases.It can cause substantial economic losses and remains a major threat to the avian industry worldwide.Phylogenetic analysis has revealed Newcastle disease virus(NDV) strains include two distinct classes(class I and class Ⅱ) within a single serotype.NDV can be detected in the wild birds and live bird markets.All class I NDV strains are lentogenic under natural conditions,however,at least there is one lentogenic class I NDV strain showed the power to become virulent under experiment conditions in the past,it is a potential threat to avian industry.Therefore,a quick method to detect NDV class I should be developed.Pair of primers and a Taq Man probe was 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 standardcurve.And then a rea1-time fluorescent quantitative RT-PCR assay was established.The method has a good linear relationship within the 106 to 101 copies,with which 1 copy/μL of the virus nucleic acid can be detected in the initial template,and has similar sensitivity with traditional virus isolation methods.The conforming rate with traditional virus isolation method was 100.0% in detecting 33 NDV isolates.The result showed that the constructed method paved the way for the rapid detection of NDV to make a distinction between class I and class I I as well as quantitative analysis for the infect degree of NDV.
Newcastle disease(ND) is one of the most serious poultry diseases.It can cause substantial economic losses and remains a major threat to the avian industry worldwide.Phylogenetic analysis has revealed Newcastle disease virus(NDV) strains include two distinct classes(class I and class Ⅱ) within a single serotype.NDV can be detected in the wild birds and live bird markets.All class I NDV strains are lentogenic under natural conditions,however,at least there is one lentogenic class I NDV strain showed the power to become virulent under experiment conditions in the past,it is a potential threat to avian industry.Therefore,a quick method to detect NDV class I should be developed.Pair of primers and a Taq Man probe was 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 standardcurve.And then a rea1-time fluorescent quantitative RT-PCR assay was established.The method has a good linear relationship within the 106 to 101 copies,with which 1 copy/μL of the virus nucleic acid can be detected in the initial template,and has similar sensitivity with traditional virus isolation methods.The conforming rate with traditional virus isolation method was 100.0% in detecting 33 NDV isolates.The result showed that the constructed method paved the way for the rapid detection of NDV to make a distinction between class I and class I I as well as quantitative analysis for the infect degree of NDV.
引文
[1]崔治中.我国家禽新城疫的流行现状[J].中国家禽,2002,24(4):426.
[2]Mark G.Wise,David L.Suarez,Bruce S.Seal,et al.Development of a real-time reverse-transcription PCR for detection of Newcastle Disease virus RNA in clinical samples[J].Journal of Clinical Microbiology,2004,42:329-338.
[3]Office of International Epizootes.Manual of standards for diagnostic tests and vaccines[M].4th ed.Paris,France:Office of International Epizootes,2000.
[4]F.Watzinger,K.Ebner,T.Lion.Detection and monitoring of virus infections by real-time PCR[J].Molecular Aspects of Medicine,2006,27:254-298.
[5]Lomniczi,B.,E.Wehmann,J.Herczeg,et al.Newcastle disease outbreaks in recent years in western Europe were caused by an old(virus isolation)and a novel genotype(VII)[J].Arch Virol,1998,143:49-64.
[6]Aldous,E.W.,D.J.Alexander.Detection and differentiation of Newcastle disease virus(avian paramyxovirus type 1)[J].Avian Pathol,2001,30:117-128.
[7]Beate M.Crossley,Sharon K.Hietala,Liu-Mei Shih,et al.High-throughput real-time RT-PCRassay to detect the exotic Newcastle Disease Virus during the California 2002-2003outbreak[J].J Vet Diagn Invest 2005,17:124-132.
[8]L.Mia Kim,Daniel J.King,David L.Suarez,et al.Characterization of Class I Newcastle Disease Virus isolates from Hong Kong Live Bird Markets and detection using real-time reverse transcription PCR[J].Journal of clinical microbiology,2007(45):1310-1314.
[9]Sheau Wei Tan,Aini Ideris,Abdul Rahman Omar,et al.2009.Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCRwith nucleocapsid gene-specific primers.Journal of Virological Methods,VIRMET10895:1-8
[10]刘晓文.2009.家鸭中弱毒新城疫病毒的分子流行病学和以弱毒为骨架表达强毒F和/或HN基因对重组病毒毒力的影响.扬州大学博士学位论文
[11]金仕强,孟春春,仇旭升等.Class I新城疫病毒概述.动物医学进展.2012,33(4):102-106
[2]Mark G.Wise,David L.Suarez,Bruce S.Seal,et al.Development of a real-time reverse-transcription PCR for detection of Newcastle Disease virus RNA in clinical samples[J].Journal of Clinical Microbiology,2004,42:329-338.
[3]Office of International Epizootes.Manual of standards for diagnostic tests and vaccines[M].4th ed.Paris,France:Office of International Epizootes,2000.
[4]F.Watzinger,K.Ebner,T.Lion.Detection and monitoring of virus infections by real-time PCR[J].Molecular Aspects of Medicine,2006,27:254-298.
[5]Lomniczi,B.,E.Wehmann,J.Herczeg,et al.Newcastle disease outbreaks in recent years in western Europe were caused by an old(virus isolation)and a novel genotype(VII)[J].Arch Virol,1998,143:49-64.
[6]Aldous,E.W.,D.J.Alexander.Detection and differentiation of Newcastle disease virus(avian paramyxovirus type 1)[J].Avian Pathol,2001,30:117-128.
[7]Beate M.Crossley,Sharon K.Hietala,Liu-Mei Shih,et al.High-throughput real-time RT-PCRassay to detect the exotic Newcastle Disease Virus during the California 2002-2003outbreak[J].J Vet Diagn Invest 2005,17:124-132.
[8]L.Mia Kim,Daniel J.King,David L.Suarez,et al.Characterization of Class I Newcastle Disease Virus isolates from Hong Kong Live Bird Markets and detection using real-time reverse transcription PCR[J].Journal of clinical microbiology,2007(45):1310-1314.
[9]Sheau Wei Tan,Aini Ideris,Abdul Rahman Omar,et al.2009.Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCRwith nucleocapsid gene-specific primers.Journal of Virological Methods,VIRMET10895:1-8
[10]刘晓文.2009.家鸭中弱毒新城疫病毒的分子流行病学和以弱毒为骨架表达强毒F和/或HN基因对重组病毒毒力的影响.扬州大学博士学位论文
[11]金仕强,孟春春,仇旭升等.Class I新城疫病毒概述.动物医学进展.2012,33(4):102-106