Rapid detection and differentiation of Newcastle disease virus isolates by a triple one-step RT-PCR

A triple one-step RT-PCR was developed to screen and differentiate virulent from avirulent Newcastle disease virus (NDV) isolates. Three sets of oligonucleotides were designed, each specific for amplifying NDV fusion protein gene-specific RNA from virulent, avirulent or all isolates respectively. The sensitivity of one-step RT-PCR was determined using viral RNA extracted from serially diluted NDV-infected allantoic fluid and found to be 10(-5) HA units. Application of one-step RT-PCR to various NDV samples, including wild-type virulent isolates and avirulent vaccine strains, demonstrated the potential for rapid identification (3-4 h) of NDV isolates as well as the differentiation of virulent from avirulent strains.     

新城疫病毒荧光RT-PCR检测方法的建立及应用

经对部分发表在NCBI里的新城疫病毒序列进行比对,在其F基因上找到1段相对保守序列,根据保守序列设计1对引物和1条TaqMan探针,以新城疫Ⅰ系疫苗株(Mukteswar株)作为标准品,通过系列条件优化、特异性、灵敏性和重复性试验,建立了一种灵敏度高、特异性强和重复性好的荧光RT-PCR检测新城疫病毒的方法.该方法能检测最低初始病毒浓度为102.2 ELD50/mL,且在108.2～102.2 ELD50/mL内Ct值与病毒浓度的对数值(lgELD50/mi)呈很好的线性关系,R2达到0.997 5;该方法对禽流感、禽痘病毒和禽大肠杆菌等禽病病原核酸无交叉反应;批间和批内重复性良好;临床样品检测中荧光RT-PCR与鸡胚分毒的符合率、诊断灵敏度和诊断特异性分别为98.5％,50.0％和98.4％.因此,荧光RT-PCR检测方法的建立为新城疫病毒检测提供了一种快速有效的方法,为新城疫病毒的研究奠定了一定的基础.     

Sensitive, semi-nested RT-PCR amplification of fusion gene sequences for the rapid detection and differentiation of Newcastle disease virus

A rapid, sensitive and specific semi-nested RT-PCR was developed to detect and differentiate virulent and avirulent strains of Newcastle disease virus (NDV). For a total of 67 NDV strains, the results obtained from the semi-nested RT-PCR were consistent with those from nucleotide sequence analysis, plaque forming assays, mean death time (MDT) measurements and intracerebral pathogenicity index (ICPI). Furthermore, 13 class I NDV strains can be characterized by the semi-nested RT-PCR approach, which was feasible by the conventional methods. The detection limit for the semi-nested RT-PCR was two plaque forming units (PFU) both for NDV strain F48E9 in allantoic fluid and for isolate APMV1/ch/ChinaND4031 in oral or cloacal swabs. In conclusion, this semi-nested RT-PCR method offers a new assay for the rapid detection and differentiation of NDVs.     

RT-PCR对新城疫病毒强弱毒株的快速鉴定

根据新城疫病毒(NDV)F蛋白基因结构的特点及强、弱毒株F蛋白基因裂解位点的序列差异设计了4条引物,并将其配成3对引物,采用RT-PCR试验对来自鸡和七彩山鸡的9株NDV野毒分离株,F48E8和LaSota 2株标准强、弱毒株进行了快速鉴定;并将RT-PCR鉴定结果与NDV常规致病性试验结果进行了比较。结果表明,RT-PCR试验能够快速将11个参试毒株区分为强毒株和弱毒株,其结果与传统致病性试验测定结果基本一致,该方法可以用于ND的快速诊断和强、弱毒株的快速鉴定。     

Differential detection of Newcastle disease virus strains by degenerate primers based RT-PCR

Degenerate primers based RT-PCR (previously described by [Avian Dis 26 (1997) 837]) has been used for the detection and differentiation of Newcastle disease (ND) viruses. Two sets of primers (A+B and A+C), with common forward primer and distinct reverse degenerate primers, designed from fusion protein gene encoding for cleavage site, could differentiate virulent and avirulent Newcastle disease viruses (NDV). Both sets of primers amplified "F" gene sequence of virulent (velogenic and mesogenic) viruses, whereas in avirulent strains, amplification was only with primer set A+C. Total 10 NDV isolates and two clinical samples including both known and unknown pathotypes, were checked. Based on amplification results 5 viruses were found to be virulent type and 6 as avirulent with one of the two clinical samples, earlier positive by RT-PCR using non-degenerate "F" gene specific primers was found negative in this study. The technique has been found to be a simple and quick for the detection and differentiation of virulent and avirulent NDV, which is important for control of the disease in the events of the outbreaks.     

用RT-PCR对新城疫病毒分离株毒力的快速鉴定

根据新城疫病毒NDV强、弱毒株在F蛋白裂解位点氨基酸组成的序列上的差异,参照有关文献,设计了三对引物A+B,A+C,A+D。引物A+B能从Lasoa,B1,I系和强毒F48E8的含毒尿囊液中扩增出362bp的核酸片段,引物A+C仅能从强毒F48E8的含毒尿囊液中扩增出254bp的片段,引物A+D仅能从Lasota,B1,I系的含毒尿囊液扩增出254bp片段。三对引物均不能从IB,IBD,AI的含毒尿囊液和正常鸡胚尿液中扩增出特异片段。从发病鸡群中分离到3株NDV毒株中均被A+C引物扩出,它们的鸡胚平均死亡时间MDT分别为51.8h、53.2h、52.6h,1日龄雏鸡脑内接种指数ICPI为1.65、1.63、1.60,证明是NDV强毒。利用RT-PCR对NDV人工感染鸡最早可于攻毒后第二天从病鸡气管中检测到NDV,其次为泄殖腔和脾、肾。利用RT-PCR对NDV毒力的鉴定与传统生物学试验对强弱毒的测定结果完全吻合。但前者可在24小时内直接对组织匀浆进行诊断,具有快速简便和敏感的特点。     

新城疫病毒通用型实时RT-PCR检测方法的建立与应用

采用TaqMan方法,经引物和探针的设计、筛选及反应条件优化,研究了检测活禽和禽产品中新城疫病毒的通用型实时RT-PCR(RRT-PCR)方法。结果显示,对12株分别为速发型、中发型、缓发型和疫苗株新城疫病毒的尿囊液倍比稀释液的检测极限在10-5~10-7之间;建立的方法与常见禽类病毒无交叉反应,特异性良好;在检测人工感染肉鸡的脏器组织、咽喉、泄殖腔拭子中病毒的灵敏度同鸡胚分离试验基本一致;弱毒疫苗免疫鸡群在免疫后14 d,应用本方法不能从咽喉、泄殖腔拭子中检测到病毒;临床样品检测表明,该方法不仅可以检出中强毒力新城疫毒株,也可检出缓发型野毒株和疫苗毒株。     

检测鸡新城疫病毒和鸡传染性支气管炎病毒双重RT-PCR的建立与初步应用

为了快速检测临床样品中新城疫病毒(NDV)和鸡传染性支气管炎病毒(IBV)的混合感染,根据NDV的F基因和IBV的N基因保守序列分别设计了2对特异性引物,优化双重RT-PCR反应条件,建立了可同时检测NDV和IBV的快速双重反转录PCR(RT-PCR)诊断方法。敏感性试验表明,此方法对NDV和IBV的RNA最小检出量分别为0.014 3和0.013 6ng;NDV的扩增片段大小为459bp,IBV的扩增片段为282bp。对其他病毒如IBDV、AIV和EDSV的检测结果均为阴性,表明此方法具有较好的特异性。对58份临床疑似样品进行检测,NDV和IBV的阳性率分别为44.82%和31.03%,NDV和IBV混合感染率为20.68%,表明本研究建立的NDV与IBV双重RT-PCR检测方法,可用于临床发病病料中NDV和IBV混合感染的快速鉴别诊断。     

Interaction between chicken lymphocytes and Newcastle disease virus

Different strains of Newcastle disease virus (NDV) were added to chicken lymphocytes and JMV-1 and MSB-1 Marek's-disease-derived tumor cell lines to determine the virus-cell interaction. NDV caused fusion and lysis of the cells, and cells supported the growth and multiplication of NDV.     

新城疫病毒单抗酶联试剂盒与RT-PCR方法检测新城疫强毒感染的比较

新城疫 ( ND)是威胁我国养禽业的头号疾病 ,病原是新城疫病毒 ( NDV)。 NDV只有一个血清型 ,其抗原性一致 ,但毒力差异很大。强毒毒株可引起易感鸡群高发病率和高死亡率的典型 ND,在免疫鸡群则引起低死亡率的非典型 ND;弱毒株不致病 ,有的弱毒株还被用作弱毒疫苗。由于弱株在自     

应用多重反转录-聚合酶链反应检测鸡新城疫病毒和传染性支气管炎病毒的研究

本文建立了一种同时检测NDV和IBV二种病原体的多重PCR技术。根据NDV和IBV的基因文库 ,设计了两对分别与NDV和IBV某段基因序列互补的引物 ,用这两对引物对同一样品中NDV和IBVRNA模板进行多重RT_PCR扩增 ,结果均同时得到了两条特异性的大小与实验设计相符的 31 0bp(NDV)和 1 72 0bp(IBV)多重的RT_PCR扩增带 ,而对其他 6种禽病病原的PCR扩增结果均为阴性 ;敏感性测定结果表明 ,该多重RT_PCR技术能同时检出 1 0pg的NDV和 1 0 0pg的IBVRNA模板。     

火龙果主要病毒RT-PCR检测方法的建立及应用

建立快速而稳定的火龙果病毒病分子检测体系,为火龙果病毒病的分子生物学鉴定提供技术支持。本文以感病的火龙果茎为研究材料,根据火龙果主要病毒病Pitaya virus X（PiVX）、Schlubergera virus X（SchVX）、Cactus virus X（CVX）及Zygocactus virus X（ZyVX）的保守序列设计特异性引物,运用RT-PCR技术进行扩增,将目的片段回收、克隆和测序。结果表明四种病毒序列与NCBI数据库里相应病毒核苷酸序列均具有较高的一致性。利用梯度稀释法依次将cDNA稀释为2、22、23、24、25、26、27倍,对各病毒灵敏度进行分析,结果显示,能检测出大部分病毒的火龙果cDNA最低浓度是45.75 ng·μL-1。应用该方法对贵州省罗甸县部分火龙果生产园内植株进行病原检测,结果显示该检测体系能特异、灵敏、稳定地检测出PiVX、SchVX、ZyVX及CVX这四种病毒。     

口蹄疫病毒TaqMan荧光定量PCR检测方法的建立

为建立检测口蹄疫病毒(FMDV)的方法,本研究根据GenBank中FMDV的2B基因序列,设计合成一对引物和一条TaqMan探针,将2B基因克隆到pBlueScriptSK(-)载体中,利用T7体外转录试剂盒制备标准品,通过优化反应条件,建立了TaqMan荧光定量PCR检测方法.结果表明,该检测方法的敏感性达到102拷贝/μL;与其它主要相关病毒均不发生交叉反应,批内和批间试验重复性的变异系数(CV)均小于3％.本研究建立的FMDV TaqMan荧光定量PCR方法对FMDV的快速检测具有重要意义.     

鸡新城疫病毒荧光RT-PCR与病毒分离方法的比较

2006年冬春季,在广州附近地区的一些竹丝鸡场、三黄鸡场、珠珍鸡场的35~50日龄鸡群相继暴发了以高死亡率、伴有呼吸道和神经症状的疫情。病鸡张口伸颈呼吸,尖叫、咳嗽,脚软,剖检病变以支气管堵塞为特征,偶见肺充血、出血、盲肠栓子、腺胃出血,病程10 d左右,死亡率20%~40%,愈后鸡群出现较多有神经症状鸡及咳嗽的鸡。我们到6个发病鸡场(A、C、D、F、G、H),现场调查流行病学、临床症状,并收集待检样本材料,同时进行新城疫病毒荧光RT-PCR检测与常规病毒分离鉴定诊断,比较两1材料与方法1.1待检病例样本包括血清样本Aa(A场发病4日龄鸡群)、C…     

Characterization of a battery of monoclonal antibodies for differentiation of Newcastle disease virus and pigeon paramyxovirus-1 strains

Twenty monoclonal antibodies (MCAs) prepared against the velogenic GB-Texas strain of Newcastle disease virus (NDV) and the type 1 pigeon paramyxovirus (PPMV-1) were characterized and examined as potential immunodiagnostic reagents. All MCAs generated were found to bind specifically, but with varying reactivity, to various NDV strains in direct binding assays. In addition, MCA 15C4 neutralized and inhibited hemagglutination (HA) of all lentogenic, mesogenic, and velogenic NDV strains tested but not the PPMV-1 strain. Antibody 10D11 also inhibited HA activity, but inhibition was more selective and limited to the mesogenic and domestic or indigenous velogenic strains of NDV. MCA 79 reacted in all serologic assays with an antigenic site common to all serotype 1 avian paramyxoviruses. Passive immunization studies involving three different neutralizing MCAs (35, 79, and 15C4) showed that enhanced, but not complete, protection against virulent NDV challenge was provided when the three MCAs were administered in combination.     

马流感病毒多重RT-PCR检测方法的建立

根据马流感病毒的M基因和HA基因的保守序列,设计了两对引物,MF和MR为通用引物,可以检测出H3N8和H7N7亚型EIV,目的片段227bp,HF和HR为H3N8亚型特异性引物,目的片段595bp。利用这两对引物,通过对多重RT-PCR扩增条件的优化,建立了快速检测鉴别H3N8亚型EIV的多重RT-PCR技术。特异性和敏感性试验结果表明,该技术可以用于EIV的快速诊断和亚型鉴定。     

A novel oligonucleotide probe for the detection of Newcastle disease virus.

An approach that possesses high specificity and broad applicability was used to obtain a DNA probe with potential diagnostic value. By utilizing synthesized oligonucleotide DNA, termed NDV probe, all 14 strains of NDV tested under high-stringency conditions were recognized in a slot-blot hybridization assay. The sequence of the NDV probe was generated from a highly conserved region of the NDV genome. No hybridization was observed with RNA isolated from other avian viruses, including avian influenza, infectious bursal disease, and infectious bronchitis. The specificity inherent in using an oligonucleotide probe offers advantages over probes obtained from cloned DNA fragments.     

RT—PCR对新城疫病毒（NDV）分离株的鉴定及其临床应用

采用新城疫病毒的通用引物PA PB、强毒引物PA PC、弱毒引物PA PD对11株贵州新城疫分离毒株与4株新城疫参考毒株进行了RT-PCR扩增.结果15株新城疫毒株均被PA PB引物扩增出359 bp的条带,F48E8、ND98、Fw、H2、P3、BY、L2、P1,P2被PA PC引物扩增出254 bp的条带,而PA PD引物未扩增出DNA条带;ND89、Lasota被PA PD引物扩增出254 bp的条带.而PA PC引物未扩增出DNA条带.采用3对引物对自然发病斗鸡脑、脾、咽喉试子、泄殖腔试子进行RT-PCR检测,确诊为斗鸡新城疫强毒感染,且与传统病毒分离与毒力鉴定的结果相一致.     

interactions between escherichia coli and Newcastle disease virus in chickens

We investigated the interaction between Newcastle disease virus (NDV) and Escherichia coli in cell cultures, embryonated eggs, and 8-wk-old chickens. We measured the interactions on the basis of bacterial adherence and NDV hemagglutination titer in chickens, chicken embryos, and chicken embryo cell culture. Depending on the inoculation order of E. coli, a significant alteration of the growth of NDV was observed in both chickens and chicken embryos. When certain strains of E. coli were given before NDV exposure, the virus titers were lowered. In chickens, the mean virus titer was significantly (P < 0.05) lowered in the crop, the proventriculus, the gizzard, and the jejunum. However, there were no significant differences (P < 0.05) between the two groups for NDV titers in the duodenum, ileum, and cecum. In chicken embryos, when E. coli serotypes O78 and O119:B14 were inoculated before NDV exposure, the mean NDV titers were significantly (P < 0.5) lowered. However, there were no significant differences (P < 0.05) in NDV titer between the two groups when E. coli serotypes O78:K80:NM and O1ab:K NM were inoculated 24 hr before NDV exposure. When NDV was given prior to E. coli exposure, NDV titer was higher in both chickens and chicken embryos. In chickens, when NDV was given 48 hr before E. coli inoculation, NDV was detected in the proventriculus, gizzard, jejunum, ileum, and cecum, whereas no virus was detected in the control groups (NDV only). In the crop, NDV was detected at a significantly (P < 0.05) higher titer in the E. coli-inoculated group when compared with the control group that received NDV alone. In chicken embryos, virus titer was significantly (P < 0.05) higher when NDV was given 24 hr before E. coli inoculation for all three NDV strains used (Ulster and V4 strains). Adherence of E. coli to chicken embryo kidney (CEK) cells was significantly higher (P < 0.05) when the CEK cells were infected first with NDV and then by E. coli. The mean bacterial count per microscopic field in NDV-uninfected monolayers was eight compared with 112 for the NDV-infected monolayers. In approximately 10% of the fields in NDV-infected monolayers, the bacteria were too numerous to count.