新城疫贵州不同分离株的蚀斑克隆与毒力鉴定
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摘要
新城疫(Newcastle Disease，ND)是由新城疫病毒引起的一种急性、高度接触性禽类传染病，被世界动物卫生组织(OIE)定为A类传染病。目前国内对于新城疫的诊断，主要是采用传统的病原分离鉴定及血凝抑制(HI)等血清学试验，虽为大家所公认，但这些方法费时、费力。本文主要开展了RT-PCR技术检测新城疫病毒和鉴别新城疫强弱毒株的研究及蚀斑克隆技术纯化NDV，为我省对新城疫的快速准确诊断奠定了技术基础。
1．应用RT-PCR试验，鉴定NDV及其强弱毒株
采用有关文献设计合成的针对NDV F蛋白的3对引物，对15株NDV进行RT-PCR试验，鉴定NDV及其强弱毒株。三对引物分别对15株NDV分离株进行鉴定，其结果与传统毒力测定结果一致，该方法可以用于ND的诊断和NDV强弱毒株的鉴定。
为了将RT-PCR及上述引物应用于新城疫的临床诊断并推广，使之具有更广泛的应用价值，我们采用该方法对贵州的13例禽类送检病例进行鉴定。结果表明：RT-PCR可直接对病料进行检测，能在6h内确诊并区分NDV强弱毒株，其病毒检出率为15.38％(2／13)，与病毒分离率15.38％(2／13)完全相符。RT-PCR应用于临床诊断具有准确、快速的特点，克服了传统方法的不足。
2．利用蚀斑克隆技术对ND毒株进行纯化
对2株NDV贵州分离株(GN、ND_(99))及2株疫苗毒株(Ⅰ系和Ⅱ系)进行挑斑纯化，结果得到12株克隆毒。经RT-PCR技术检测，7株为弱毒，形成蚀斑的大小在0.3～1.0mm；5株为强毒，其中4株形成蚀斑的大小在1.0～2.0mm，1株为形成蚀斑的直径为0.4mm的红色蚀斑；在胰酶存在的情况下，低毒力和中等毒力的NDV在鸡胚成纤维细胞上都能形成蚀斑，所形成蚀斑的大小与病毒毒力具有相关性。
3．新城疫毒株毒力谱的构建
通过传统毒力指数测定结果和针对HN蛋白单克隆抗体的分群研究，结合RT-PCR对NDV贵州分离株的鉴定，综合评价不同现场流行毒株的毒力，构建新城疫病毒分离物的毒力谱如下：
F_(48)E_8＞FW＞BY＞H_2＞ND_(98)＞P_2＞P_1＞P_3＞L_2＞Ⅰ系＞GN＞Ⅱ系＞Ⅳ系＞ND_(99)＞ND_(89)
Newcastle disease (ND) is an acute, highly contiguous infectious disease caused by Newcastle disease virus(NDV). Newcastle disease virus is a member of Rubulaviru, Paramyxovirinae, Paramyxoviridae, and a negative RNA virus. It exists in all tissues, apparatus, body fluid, secretion and excrement.
In this paper, the detection of Newcastle disease virus by RT-PCR and virulence determination of different NDV isolates in Guizhou province were carried out. All these improved the detection technology for detecting Newcastle disease virus quickly and accurately.
1. The detection by RT-PCR and virulent determination on NDV
On the basis of difference in compositions and sequence of aminoacidat the cleavagesit of F protein of virulent and non-virulent strains of Newcastle disease virus (NDV) three primer pairs A+B,A+C and A+D were designed according to relevant references. The virulence of NDV identified by RT-PCR accorded with those by conventionally biological methods. Furthermore the method of RT-PCR to diagnose ND have sensitive, simple and rapid.
RT-PCR was used in detection of clinical case; the results confirmed the results of serology, such as hemagglutination test and hemagglutination inhibition test. The results showed that RT-PCR can be used to determine cases which caused by Newcastle disease virus and determine the virulent in 6 hours; the detection rate was higher than isolation rate with 53.84%. compared with RT-PCR, the progress of traditional methods such as MDT, ICPI and IVPI were trouble and tedious, and we need about 7-10 days to wait the results, at the same time, the possibility cross-contamination still exist in operative procedure. In this experience, RT-PCR was used to avoid the shortcomings effectively.
2. Purified of NDV strains by plaque clone technology.
Purified of 2 NDV Guizhou isolated strains (GN, ND_(99)) and 2 vaccine strains by plaque clone technology. 12 purified strains were received. The results of RT-PCR detected showed: 7 are the mesogenic virus, and the size of the plaque which they formed was 0.3-1.0 mm; 5 are the Velogenic virus, the plaque size was 1.0-2.0 mm, in which I/1 was 0.4 mm red plaque. In the non-pancreas enzyme or in the magnesium ion situation, low virulent NDV becomes on CEF not to be able to form the plaque, the full strength and medium can form plaques.
3. The constructed of virulent spectrum.
According to the determination results of conventional virulent index, and in view of the HN protein monoclonal antibody, and RT-PCR to appraisal the NDV strains, the quality synthetic evaluation different scene popular virulent, to construct the Newcastle disease virus isolation virulent spectrum. The quality synthetic evaluation three methods to the different NDV separation virulent determination result as follow:F_(48)E_8 >FW>BY> H_2>ND_(98)> P_2> P_1> P_3>L_2>GN> II > IV > ND_(99) > ND_(89)
1．应用RT-PCR试验，鉴定NDV及其强弱毒株
采用有关文献设计合成的针对NDV F蛋白的3对引物，对15株NDV进行RT-PCR试验，鉴定NDV及其强弱毒株。三对引物分别对15株NDV分离株进行鉴定，其结果与传统毒力测定结果一致，该方法可以用于ND的诊断和NDV强弱毒株的鉴定。
为了将RT-PCR及上述引物应用于新城疫的临床诊断并推广，使之具有更广泛的应用价值，我们采用该方法对贵州的13例禽类送检病例进行鉴定。结果表明：RT-PCR可直接对病料进行检测，能在6h内确诊并区分NDV强弱毒株，其病毒检出率为15.38％(2／13)，与病毒分离率15.38％(2／13)完全相符。RT-PCR应用于临床诊断具有准确、快速的特点，克服了传统方法的不足。
2．利用蚀斑克隆技术对ND毒株进行纯化
对2株NDV贵州分离株(GN、ND_(99))及2株疫苗毒株(Ⅰ系和Ⅱ系)进行挑斑纯化，结果得到12株克隆毒。经RT-PCR技术检测，7株为弱毒，形成蚀斑的大小在0.3～1.0mm；5株为强毒，其中4株形成蚀斑的大小在1.0～2.0mm，1株为形成蚀斑的直径为0.4mm的红色蚀斑；在胰酶存在的情况下，低毒力和中等毒力的NDV在鸡胚成纤维细胞上都能形成蚀斑，所形成蚀斑的大小与病毒毒力具有相关性。
3．新城疫毒株毒力谱的构建
通过传统毒力指数测定结果和针对HN蛋白单克隆抗体的分群研究，结合RT-PCR对NDV贵州分离株的鉴定，综合评价不同现场流行毒株的毒力，构建新城疫病毒分离物的毒力谱如下：
F_(48)E_8＞FW＞BY＞H_2＞ND_(98)＞P_2＞P_1＞P_3＞L_2＞Ⅰ系＞GN＞Ⅱ系＞Ⅳ系＞ND_(99)＞ND_(89)
Newcastle disease (ND) is an acute, highly contiguous infectious disease caused by Newcastle disease virus(NDV). Newcastle disease virus is a member of Rubulaviru, Paramyxovirinae, Paramyxoviridae, and a negative RNA virus. It exists in all tissues, apparatus, body fluid, secretion and excrement.
In this paper, the detection of Newcastle disease virus by RT-PCR and virulence determination of different NDV isolates in Guizhou province were carried out. All these improved the detection technology for detecting Newcastle disease virus quickly and accurately.
1. The detection by RT-PCR and virulent determination on NDV
On the basis of difference in compositions and sequence of aminoacidat the cleavagesit of F protein of virulent and non-virulent strains of Newcastle disease virus (NDV) three primer pairs A+B,A+C and A+D were designed according to relevant references. The virulence of NDV identified by RT-PCR accorded with those by conventionally biological methods. Furthermore the method of RT-PCR to diagnose ND have sensitive, simple and rapid.
RT-PCR was used in detection of clinical case; the results confirmed the results of serology, such as hemagglutination test and hemagglutination inhibition test. The results showed that RT-PCR can be used to determine cases which caused by Newcastle disease virus and determine the virulent in 6 hours; the detection rate was higher than isolation rate with 53.84%. compared with RT-PCR, the progress of traditional methods such as MDT, ICPI and IVPI were trouble and tedious, and we need about 7-10 days to wait the results, at the same time, the possibility cross-contamination still exist in operative procedure. In this experience, RT-PCR was used to avoid the shortcomings effectively.
2. Purified of NDV strains by plaque clone technology.
Purified of 2 NDV Guizhou isolated strains (GN, ND_(99)) and 2 vaccine strains by plaque clone technology. 12 purified strains were received. The results of RT-PCR detected showed: 7 are the mesogenic virus, and the size of the plaque which they formed was 0.3-1.0 mm; 5 are the Velogenic virus, the plaque size was 1.0-2.0 mm, in which I/1 was 0.4 mm red plaque. In the non-pancreas enzyme or in the magnesium ion situation, low virulent NDV becomes on CEF not to be able to form the plaque, the full strength and medium can form plaques.
3. The constructed of virulent spectrum.
According to the determination results of conventional virulent index, and in view of the HN protein monoclonal antibody, and RT-PCR to appraisal the NDV strains, the quality synthetic evaluation different scene popular virulent, to construct the Newcastle disease virus isolation virulent spectrum. The quality synthetic evaluation three methods to the different NDV separation virulent determination result as follow:F_(48)E_8 >FW>BY> H_2>ND_(98)> P_2> P_1> P_3>L_2>GN> II > IV > ND_(99) > ND_(89)
引文
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