Real-time PCR方法和PCR方法检测虾白斑综合征病毒

由白斑综合征病毒引起的虾白斑综合征是国际兽医局（OIE）规定的必须上报的水生动物二类疫病。本研究首先通过设计新的PCR引物，提高了PCR检测白斑综合征病毒的阳性检出率。为进一步提高白斑综合征病毒检测的灵敏度，本研究采用TaqMan探针技术建立了快速检测白斑综合征病毒的real-time PCR方法，通过与常规PCR方法比较，证实其检测灵敏度显著提高。     

鸡毒支原体强弱毒株荧光定量PCR鉴别检测方法的建立

为建立一种能鉴别鸡毒支原体(MG)强、弱毒株的快速检测方法,本研究根据GenBank中MG强毒株和弱毒株的基因组序列,选取特异性保守区序列设计了2对引物和2奈探针,分别用于强弱毒株和弱毒株的检测,优化反应条件,建立了能区分MG强、弱毒株的荧光定量PCR检测方法.该法特异性强,对鸡常见呼吸道病原体的反应均为阴性;灵敏度高,可检测到100拷贝/μL的模板;稳定性好,批内和批间试验Ct值的变异系数小.本研究建立的MG强、弱毒鉴别检测方法简便、快捷,为该病的防控与净化提供新方法、新思路.     

禽网状内皮组织增殖病病毒PCR及RT-PCR检测方法的研究和p30主要抗原域的原核表达

根据MONA M．ALY发表的一对引物，5’-CATACTGGAGCCAATGGTGTAAA GGGCAG A-3’（上游引物），5’-AATGTTGTAGCGAAGTACT-3’（下游引物），上游引物位于REV—SNV毒株LTR序列上游237bp到267bp，下游引物在499bp到517bp之间，扩增产物总长为281bp。以REV—T株感染的鸡胚成纤维细胞DNA作为PCR扩增模板，进行PCR扩增，提取病毒RNA进行RT—PCR，这两种方法均获得了禽网状内皮增生症T株的部分LTR序列，将其克隆入pGEM—TEasy克隆载体中，经测序，证实为禽网状内皮增生症T株的部分LTR序列，初步建立了REV的PCR和RT-PCR检测方法。人工合成了REVp30的主要抗原域，用构建成功的表达禽网状内皮增生症p30主要抗原域的原核表达载体pET—p30，转化大肠杆菌BL21，挑取阳性克隆培养，IPTG诱导后裂解菌体作SDS—PAGE分析，出现大小约25Ku的表达产物即融合蛋白TrxA—p30主要抗原域，经Western blot验证融合蛋白具有REV特异的反应原性。采用常规表达条件：LB培养基，37℃培养，待菌生长至OD600为0．4h～0．6h。用终浓度为0．2mmol／L～0．8mmol／L的IPTG进行诱导，于37℃振荡培养4h～5h，均能高效表达。本研究为建立REV抗体的ELISA检测方法打下基础。     

套式PCR和实时荧光PCR在检测胸膜肺炎放线杆菌上的应用

应用胸膜肺炎放线杆菌套式PCR和实时荧光PCR检测方法分别检测了不同来源的猪鼻拭子、肺、扁桃体等494份样品。套式PCR 77份阳性,实时荧光PCR 62份阳性,前者的阳性完全覆盖了后者的阳性,两者符合率为97%(479/494)。2种方法均可应用于猪传染性胸膜肺炎的诊断和监测。     

实时荧光PCR检测牛边缘无浆体方法的建立及应用

根据牛边缘无浆体表面蛋白4的保守基因序列设计特异引物AMOC9/AMOC5、AMOC10/AMOC12和特异性探针MP,首次建立了牛边缘无浆体的实时荧光PCR检测方法,检测DNA的最低限度为200 fg。对中央无浆体、绵羊无浆体、牛巴贝斯虫、双芽巴贝斯虫、羊莫氏巴贝斯虫、山羊泰勒虫、温氏附红细胞体、东方巴贝斯虫、刚地弓形虫和伊氏锥虫进行检测,无荧光检测信号。本研究用所建立的方法检测采自江苏和哈尔滨的180份抗凝血（奶牛和肉牛）,其阳性率为8.9%。结果表明,建立的实时荧光PCR检测牛边缘无浆体的方法具有较高的特异性和敏感性,可用于牛边缘无浆体病的流行病学调查、检疫和监测。     

实时荧光PCR和常规PCR方法检测饲料中鸡源性成分

根据鸡线粒体DNA序列,设计并筛选了鸡源性特异引物及探针,建立了饲料中鸡源性成分的常规PCR和实时荧光PCR检测方法,结果表明常规PCR方法最低可以从饲料中检出0.1%的鸡源性成分,实时荧光PCR方法最低可以从饲料中检出0.001%的鸡源性成分。该方法具有很高的特异性和灵敏性,可以作为鸡源性成分鉴别检测的常规方法。     

Identification and characterization of fowlpox virus strains using monoclonal antibodies.

The use of 2 monoclonal antibodies (MAbs), P1D9 and P2D4, which recognize different fowlpox virus (FPV) antigens, for the identification and characterization of FPV strains was evaluated. Initially, the MAbs were used in conjunction with a dot blot assay that enabled FPV to be differentiated from the avian herpesvirus, infectious laryngotracheitis virus. Confirmation of the specificity of these MAbs was provided by the demonstration that only FPV antigens were recognized by a combination of both antibodies when used for immunoblotting proteins contained in various avipoxviruses. Later, an antigenic characterization of 11 FPV field isolates, 6 FPV vaccine strains, and 3 pigeonpox virus vaccines was performed by Western blotting with the individual MAbs. Whereas MAb P2D4 consistently recognized a protein with an apparent molecular weight of 60 kD, there was variability in the size of the antigen that was immunoreactive with the other MAb. For example, MAb P1D9 recognized an antigen of apparent molecular weight of 46 kD in all vaccine strains except 2 of FPV origin. In these exceptions, either only a 39-kD or both a 42- and 46-kD protein were immunoreactive. As for the field isolates, a 39-kD antigen was recognized in 8 of them, whereas a 42-kD antigen was detected in the remaining 3. Therefore, the more extensive immunoblotting technique may facilitate FPV strain differentiation, whereas routine diagnosis of fowlpox could be accomplished by using the MAb-based dot blot assay.     

Construction and characterization of a fowlpox virus field isolate whose genome lacks reticuloendotheliosis provirus nucleotide sequences

Fowlpox virus (FWPV) has been isolated from vaccinated chicken flocks during subsequent fowlpox outbreaks that were characterized by a high degree of mortality and significant economic losses. This inability of current vaccines to induce adequate immunity in poultry could be reflective of an antigenic and/or biologic distinctiveness of FWPV field isolates. In this regard, whereas an infectious reticuloendotheliosis virus (REV) provirus is present in the majority of the field viruses' genomes, only remnants of REV long terminal repeats (LTR) have been retained in the DNAs of each vaccine strain. Although it has not been demonstrated whether the partial LTRs can provide an avenue for FWPV to reacquire the REV provirus by homologous recombination, utilizing viruses of which genomes lack any known integrated retroviral sequences could resolve concern over this issue. Therefore, such an entity was created by genetically modifying a recently isolated field strain of FWPV. This selection, in lieu of a commercial vaccine virus, as the progenitor was based on the probability that a virus circulating in the environment would be more antigenically similar to others in this locale and thus might be a better candidate for vaccine development. A comparison in vivo of the pathogenic traits of the parental wild-type field isolate, its genetically modified progeny, and a rescue mutant in whose genome the REV provirus was inserted at its previous location, indicated that elimination of the provirus sequence correlated with reduced virulence. However, even with elimination of the parasitic REV, the modified FWPV was still slightly more invasive than a commercial vaccine virus. Interestingly, both types of attenuated FWPV elicited a similar degree of antibody production in inoculated chickens and afforded them protection against a subsequent challenge by a field virus, the origin of which was temporally and geographically distinct from that of the progenitor strain. Due to its antigenicity being retained despite a decrease in virulence, this REV-less FWPV could potentially be developed as a vaccine against fowlpox.     

鸡痘病毒分离株及疫苗株整合网状内皮组织增生病病毒的基因分析

为了解山东地区鸡痘病毒(FPV)野毒流行株整合禽网状内皮组织增生病病毒(REV)前病毒的情况,同时比较FPV分离株和弱毒疫苗株整合REV前病毒序列的差异,本研究对山东不同地区的14个FPV分离株和国内外不同厂家的5个FPV疫苗株进行了REV前病毒整合区基因PCR扩增、核苷酸序列测定及基因分析.结果表明14个FPV分离株均整合了几乎全长的REV前病毒序列,这些序列与美国分离的FPV整合株AF246698的整合序列同源性最高,与REV标准株同源性较低;疫苗株均仅整合部分或完整的REV-LTR序列,但国产与进口疫苗株的整合序列不同,国产疫苗整合的LTR片段相对较小.研究表明所分离的山东FPV分离株均整合全长REV序列,疫苗株仅整合REV-LTR序列.然而FPV整合病毒株的致病特性、整合疫苗的安全性及对整合FPV野毒的免疫保护作用有待于进一步研究.     

荧光定量RT-PCR法对犬瘟热病毒PS株感染犬血清和粪便中病毒的检测

为研究犬人工感染犬瘟热病毒(CDV)后病毒在血清和粪便中的含量及变化,本实验根据CDV核衣壳蛋白(NP)基因序列设计一对特异引物,扩增NP基因.并以NP基因重组质粒作为阳性标准品,建立检测CDV的SYBR GreenⅠ荧光定量RT-PCR方法.结果表明,该方法102拷贝～109拷贝范围内具有良好的线性关系,相关系数为R2=0.998,扩增效率为E=98.3％,敏感度高,最低检测限为6.1拷贝/μL,重复性检测变异系数低于2.62％.该方法与常规RT-PCR方法比较,其敏感性提高102倍.两只人工感染CDV PS强毒株的犬,分别于接种后17d、19d发病死亡,采用荧光定量RT-PCR方法对人工感染犬的血液和拭子液中的病毒核酸栽量进行定量检测以及对收集的22份临床样本拭子液进行检测,均检测到CDV.本研究结果表明,该方法可以用于CDV核酸定量检测,为该病毒的致病机理及病毒传播途径的研究提供了一种快速和敏感的检测手段.     

区别伪狂犬病病毒野毒株和gE-疫苗株的gE-MGB-TaqMan PCR方法的建立

本实验通过设计针对伪狂犬病病毒gE基因的引物和MGB(Minor groove binder oligodeoxynucleotide conjugate,MGB-ODN)TaqMan探针,结合ABI PE7700荧光定量PCR仪器系统,建立了一种能区别伪狂犬病病毒野毒株和gE-疫苗株的快速检测方法gE-MGB-TaqMan PCR。实验表明,该方法可检测出最低43拷贝的gE基因和10~4倍稀释的伪狂犬病病毒Fa株DNA,与微量血清中和结合MTT比色法相比,灵敏度和特异性一致,检测时间仅为后者的1/10,操作比后者更为简单。特异性和重复性试验表明:gE-MGB-TaqMan PCR特异性和重复性好。该方法以闭管的模式操作,减少了各步骤污染的可能性,整个检测少于3 h。     

实时荧光定量PCR检测猪细小病毒

根据已报道的猪细小病毒基因组序列,设计并合成了引物和探针,从猪细小病毒(PPV)感染的细胞中提取DNA,经PCR扩增,产物纯化后与pGEM-T-easy连接,转化大肠杆菌JM109,筛选后得到重组标准品质粒,对重组标准品质粒进行PCR和测序鉴定,表明目的片段已经成功克隆.将104～108拷贝反应的重组标准品质粒进行荧光定量PCR,系统自动分析软件显示Ct值与标准品浓度的对数之间存在良好的线性关系.动力学曲线分析表明,在该反应体系和反应条件下,标准曲线的灵敏度为102拷贝.本方法的建立为猪细小病毒感染的早期诊断奠定了基础.     

Detection of fowlpox virus DNA by in situ hybridization using a biotinylated probe

In situ hybridization was applied to detect fowlpox virus (FPV) DNA in formalin-fixed paraffin-embedded sections of the skin from infected chickens by using a biotinylated probe and a streptavidin-alkalinephosphatase conjugate. The immunohistochemical examination was applied to compare the distribution of the FPV DNA to that of related antigenic protein in serial sections. In the infected epithelial cells, FPV DNA was detected in cytoplasmic inclusion bodies and in the rest of cytoplasm. Likewise, immunohistochemical examination revealed the virus antigen in cytoplasm. Ultrastructurally, virions were observed in the cytoplasmic inclusion bodies, and immature virus particles were in the rest of the cytoplasm. The study proved restricted distribution of FPV DNA in the cytoplasm.     

鸡白痢与鸡伤寒沙门菌双重PCR方法的建立和初步应用

分析不同血清型/生物型沙门菌全基因组序列筛选出鸡白痢和鸡伤寒沙门菌特异性基因组序列,设计两对引物,建立双重PCR方法鉴别检测鸡白痢和鸡伤寒沙门菌不同生物型,并进行初步的临床应用。双重PCR方法结果显示,鸡白痢沙门菌显示417 bp条带,鸡伤寒沙门菌显示417 bp和636 bp两个条带,而阴性对照未出现条带,与预期设计相符。双重PCR体系对56株不同血清型沙门菌鉴定结果与细菌学分离的血清型鉴定结果完全一致,说明本试验建立的双重PCR体系特异性良好,应用上述方法检测鸡场疑似20份临床样本,结果发现8株鸡白痢沙门菌阳性,1株鸡伤寒沙门菌阳性。上述结果表明,已建立双重PCR方法特异性检测鸡白痢和鸡伤寒沙门菌。本试验为鸡白痢和鸡伤寒不同生物型沙门菌的检测提供了一种简洁、敏感、特异的新方法。     

Detection and genotypic differentiation of Campylobacter jejuni and Campylobacter coli strains from laying hens by multiplex PCR and fla-typing

In total, 26 Campylobacter (C.) strains, isolated from liver, spleen, caecal or jejunal content of laying hens from different flocks were examined. In these flocks a drop in egg production, an increasing mortality and livers with whitish-grey lesions as post-mortem finding were observed. Suspected Campylobacter colonies were differentiated using a modified m-PCR in 13 Campylobacter jejuni and 13 Campylobacter coli strains. All isolates were characterised by typing of the flaA and flaB gene each with two restriction enzymes. To compare the four different profiles for all strains an artificial “fla-type” was generated. Different and identical fla-types of C. jejuni and C. coli were recovered from both intestinal and extra-intestinal organs of the laying hens and even from individual birds. One significant observation is that some fla-types of C. jejuni or C. coli were detected in intestinal and systemic sites but not all fla-types of both species appeared to be equally able to invade internal organs.     

Detection of Chlamydophila psittaci by using SYBR green real-time PCR

Chlamydophila psittaci is the causative agent of human psittacosis and avian chlamydiosis. This zoonotic pathogen is frequently transmitted from infected birds to humans. Therefore proper and rapid detection of C. psittaci in birds is important to control this disease. We developed a method for detecting C. psittaci by using SYBR Green Real-time PCR based on targeting the cysteine-rich protein gene (envB) of C. psittaci. This one step procedure was highly sensitive and rapid for detection and quantification of C. psittaci from fecal samples. This assay was also able to detect other zoonotic Chlamydophila species such as C. abortus and C. felis. The assay is well suited for use as a routine detection method in veterinary medicine.     

Detection and genotypic differentiation of Campylobacter jejuni and Campylobacter coli strains from laying hens by multiplex PCR and fla-typing

In total, 26 Campylobacter (C.) strains, isolated from liver, spleen, caecal or jejunal content of laying hens from different flocks were examined. In these flocks a drop in egg production, an increasing mortality and livers with whitish-grey lesions as post-mortem finding were observed. Suspected Campylobacter colonies were differentiated using a modified m-PCR in 13 Campylobacter jejuni and 13 Campylobacter coli strains. All isolates were characterised by typing of the flaA and flaB gene each with two restriction enzymes. To compare the four different profiles for all strains an artificial “fla-type” was generated. Different and identical fla-types of C. jejuni and C. coli were recovered from both intestinal and extra-intestinal organs of the laying hens and even from individual birds. One significant observation is that some fla-types of C. jejuni or C. coli were detected in intestinal and systemic sites but not all fla-types of both species appeared to be equally able to invade internal organs.     

实时荧光定量RT-PCR方法检测禽流感病毒

根据禽流感病毒(Avian influenza virus,AIV)M基因上的保守序列,合成引物和荧光标记探针,以阳性AIVM基因质粒为标准品做标准曲线,建立了荧光定量逆转录聚合酶链反应(RRT-PCR)检测方法。结果表明,本试验建立的标准曲线循环阈值(Ct值)与模板浓度具有良好的线性关系,相关系数为0.999,灵敏度约为5拷贝/μL,相当于5个AIV颗粒,对新城疫病毒和其他禽病病毒无交叉反应,特异性好、重复性佳,为AIV检测提供了一种特异、敏感、快速的定量检测方法。对500份临床泄殖腔棉拭样品的检测,其结果阳性?阴性数与经典病毒分离方法符合率分别为91.2%?99.4%。在AIV临床样品筛检、流行病学监测等方面显示良好的应用前景。     

应用改良阻断ELISA检测禽网状内皮组织增殖病血清抗体

应用禽网状内皮组织增殖病病毒纯化抗原和抗ＲＥＶ单克隆抗体建立了改良阻断ＥＬＩＳＡ用于鸡血清中ＲＥＶ抗体检测，并对北京地区鸡群中随机采样的３６份血清样本进行了检测，阳性率为５．６％。与间接ＥＬＳＩＡ的检测结果进行了统计学比较，两种方法的阳性率无显著差异。结果表明本试验所建立的改良阻断ＥＬＩＳＡ可以用于鸡群ＲＥＶ感染的血清学调查。