地高辛标记的核酸探针检测EDS病毒的研究

地高辛标记的核酸探针检测ＥＤＳ病毒的研究王雪敏（邯郸农业高等专科学校牧医系河北永年０５７１５０）郑明球蔡宝祥陈溥言（南京农业大学动物医学院１材料与方法１．１地高辛标记的ＥＤＳ病毒核酸探针制备将ＥＤＳ病毒ＮＥ４株（南京农大传染病教研室提供）鸭胚尿囊液（...     

地高辛标记核酸探针检测牛粘膜病病毒

本课题建立了地高辛标记核酸探针检测牛病毒性腹泻－粘膜病病毒（ＢＶＤＶ）的诊断方法。根据ＢＶＤＶ基因序列的结构特点,在编码非结构蛋白ｐ１２５高度保守基因区内,设计合成一对特异性引物。以ＰＣＲ技术从ＢＶＤＶ基因重组质粒中扩增出了ＢＶＤＶ特异的、保守的４００ｂｐ核苷酸片段,经纯化后,地高辛标记,制备牛病毒性腹泻病毒的特异性核酸探针。通过检测ＢＶＤＶ细胞培养物、相关病毒及核酸载体和ＢＶＤＶ细胞培养物的总Ｒ     

地高辛标记核酸探针检测鸭瘟病毒

鸭瘟是鸭、鹅和其他雁形日禽类的一种急性、热性、啦血性传染病．特，征足流行广泛，传播迅速．发病率高和死亡率大。由于鸭瘟引起死亡、淘汰和产蛋下降，给发病地区的商品鸭和产蛋鸭造成很大的经济损失，目前．国内检症、诊断该病常用的方法有病毒分离、琼脂扩散试骑、酶联免疫吸附试验等。国内尚未见地高辛标记核酸探针检测鸭瘟病毒的报道。     

用地高辛标记核酸探针检测鹅细小病毒的研究

从带有鹅细小病毒(GPV)NSI基因的重组质粒pMD18T-NS1用限制性内切酶EcoRI和BamH1双酶切回收1880bp大小片段,并制备出地高辛标记的GPV核酸探针。其标记效率达到0．01pg／μl。特异性检测结果表明,该探针能与GPV不同毒株核酸发生特异性杂交,而与对照的DPV、GPPV等病毒的核酸杂交反应均为阴性;敏感性检测结果表明,该探针对GPV的最低检出量为0．0224pg。该探针对不同方法处理的GPV感染病料进行检测,均出现杂交阳性。表明所研制的标记探针用于GPV的检测足可行的。     

用地高辛标记核酸探针检测猪细小病毒的研究

根据猪细小病毒(PPV)基因组序列,设计引物克隆了PPV YK株保守序列NS1基因,将NS1基因纯化,利用地高辛标记制备了NS1基因PCR产物探针和克隆NS1探针,两种核酸探针的标记都达到了0.1 pg/μL。特异性试验结果表明,这两种探针都能与PPV DNA发生特异的阳性杂交,而与对照的PRV、PCV、PRRSV和HCV的核酸杂交呈阴性。敏感性试验结果表明,克隆NS1探针敏感度为0.5 pg/μL,NS1基因PCR产物探针敏感度为1 pg/μL。综上所述,这两种DIG标记探针特异性强,敏感性高,可用于PPV的检测。     

地高辛标记猪细小病毒核酸探针的研制

利用ＰＳｔⅠ和ＨｉｎｄⅢ双酶切及低熔点琼脂糖凝胶电泳技术，从重组质粒ＰＵＰ中回收３．０ｋｂ猪细小病毒ＤＮＡ片段。以地高辛（Ｄｉｇｏｘｉｇｅｎｉｎ）标记后制备探针。运用该探针对作者分离并鉴定的７株猪细小病毒及ＰＰＶ－ＢＭ１株、四川株、Ｚ株、广西株、天津株进行打点杂交，并以具有相同临床症状的猪病毒性疾病的猪瘟病毒、日本乙型脑炎病毒、伪狂犬病病毒以及ＰＵＰ质粒、ＰＫ－１５为对照。结果探针与猪细小病毒ＤＮＡ、ＰＵＰ质粒的杂交呈阳性反应，而对照病毒、ＰＫ－１５呈阴性反应，探针的最低检出限量为４０ｐｇＤＮＡ。结果表明，该探针具有特异性强、敏感性高，可用于猪细小病毒的检测     

地高辛标记C15A核酸探针检测牛巴贝斯虫

为了提高牛巴贝斯虫核酸探针的检测性能,制备了地高辛标记的牛巴贝斯虫Ｃ１５Ａ核酸探针。该探针检测牛巴贝斯虫ＤＮＡ的灵敏度为３２ｐｇ,检测探针ＤＮＡ的灵敏度为０．１ｐｇ;检测其他对照血液原虫（双芽巴贝斯虫、边缘无浆体、瑟氏泰勒虫、伊氏锥虫、卵圆巴贝斯虫）和牛血细胞的ＤＮＡ,均未出现非特异性反应。与光敏生物素标记的牛巴贝斯虫Ｃ１５Ａ核酸探针比较,光敏生物素标记探针能检出１０％带虫血１２．５μＬ,而地高辛标记探针能检出１０％带虫血０．０１５μＬ,且杂交背景浅,显色深。     

探针检测鸭黄病毒的地高辛标记DNA的制备与应用

利用RT-PCR方法扩增鸭黄病毒的NS3基因406bp的特异性片段,回收并纯化PCR产物,用地高辛标记,制备核酸探针。特异性试验结果表明,该探针仅与鸭黄病毒的核酸特异性杂交,而与鸭瘟病毒、H9N2禽流感病毒、新城疫病毒、传染性法氏囊病病毒、减蛋综合征病毒的核酸杂交均为阴性。敏感性试验表明,该探针对鸭黄病毒的RNA最低检出限量为100μg/L。对疑似黄病毒感染鸭的肝脏、肺脏、脾脏、输卵管、卵泡膜和泄殖腔棉拭子进行检测,以卵泡膜的检出率最高。该研究为鸭黄病毒感染的诊断和流行病学调查提供了一种可靠的方法。     

应用PCR技术检测鹅细小病毒

根据鹅细小病毒GPVH1株结构蛋白VP1与VP3编码基因非重叠核苷酸序列设计了一对引物GPR1 /GPR2 ,用这对引物对感染器官内的GPR和接种鹅胚增殖的GPV进行PCR扩增 ,其结果引物GRP1 /GPR2能特异性地扩增GPVVP1 VP3区段核苷酸序列 ,扩增出与设计核苷酸片段大小相符的 0 6kb的序列 ,而对照的鹅副粘病毒cDNA及鸭瘟病毒 (DPR)DNA对照组均出现阴性结果。     

禽流感RT-PCR诊断法的建立

根据禽流感病毒（ＡＩＶ）Ａ／Ｃｈｉｃｋｅｎ／Ｂｒｅｓｌｉｎ／１９０２（Ｈ７Ｎ７）株的血凝素（ＨＡ）基因参考序列设计合成一对Ｈ７ＨＡ特异引物,并应用ＳＤＳ－蛋白酶Ｋ法提取病毒ＲＮＡ,建立了逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）检测ＡＩＶＲＮＡ的方法。应用此法对鸡胚培养的ＡＩＶ尿囊液、ＳＰＦ感染鸡粪便进行了检测,并与琼脂扩散试验和电镜技术作了对比。特异性试验以新城疫病毒（ＮＤＶ）、传染性法氏囊病病毒（ＩＢＤＶ）、传染性支气管炎病毒（ＩＢＶ）、减蛋综合征病毒（ＥＤＳ－７６Ｖ）等的感染鸡胚尿囊液作为对照。结果表明,ＡＩＶ尿囊液ＲＴ－ＰＣＲ全部阳性,ＳＰＦ感染鸡粪便８７份,ＲＴ－ＰＣＲ检出６９份阳性,样品处理浓缩后琼脂扩散检出１１份,电镜检测了２３份样品,仅检出２份阳性。非特异性对照样品经ＲＴ－ＰＣＲ检测全部阴性。将ＡＩＶ感染鸡胚尿囊液作１０倍系列连续稀释,可检出稀释度为１０－２的病毒尿囊液。ＲＴ－ＰＣＲ最早检出时间为感染后第３天,而琼扩和电镜均是７天。该法具有高度的特异性和灵敏性,且节约时间（只需８小时左右）,可从分子水平上对ＡＩＶ进行早期快速诊断和流行病学调查     

Application of the polymerase chain reaction (PCR) in veterinary diagnostic virology

The polymerase chain reaction has become an important diagnostic tool for the veterinary virologist. Conventional methods for detecting viral diseases can be laborious or ineffective. In many cases PCR can provide a rapid and accurate test. In this article we explain the basic principles of PCR and supply a reference list of its uses in diagnostic veterinary virology.Abbreviations BLV bovine leukaemia virus - BVDV bovine viral diarrhoea virus - DNA deoxyribonucleic acid - ds double-stranded - ELISA enzyme-linked immunosorbent assay - PCR polymerase chain reaction - RNA ribonucleic acid - ss single-stranded - TK thymidine kinase     

Use of serology and polymerase chain reaction for the rapid eradication of small ruminant lentivirus infections from a sheep flock: A case report

A SRLV-free sheep flock incurred infection which led to an SRLV infection rate of over 50% of the ewes (34/64) within a 30 months period, indicating that environmental conditions were favourable to transmission. An intensive regimen of sampling at short intervals and testing for SRLV antibodies and proviral DNA combined with strict management was implemented for the entire flock, lambs and yearlings included. This resulted in eradication of the infection within two testing and culling rounds with a 3 months interval. The additional value of the proviral DNA detection by PCR in identifying infected animals was clear in that nine infected animals were found that would have been missed if tested by serology alone. PCR also saved two lambs from being culled; they were sero-positive probably due to maternal antibodies, but not infected.     

PCR技术检测饲料中沙门氏菌的应用研究

用聚合酶链反应(PCR)技术检测饲料中沙门氏菌,对已知被沙门氏菌污染的动物饲料培养物均能检出阳性,说明本方法对检测饲料中沙门氏菌具有特异性高、灵敏、快速等特点,适用于快速、准确地检测饲料中沙门氏菌的需要。     

RT-PCR快速诊断禽流感

根据禽流感病毒ＮＰ基因的序列分析结果,设计了一对ＮＰ基因特异的引物。采用该对引物,不经病毒分离,直接从禽流感病毒感染鸡的气管、泄殖腔棉拭子和组织样品中提取核酸, ＲＴ～ＰＣＲ可以扩增出 ３２６ｂｐ的 ＮＰ基因片段。采用该技术对１４个亚型禽流感病毒标准参考株,４个亚型１２株国内分离野毒株,ＲＴ－ＰＣＲ检测的结果都呈阳性;对新城疫病毒、传染性法氏囊病毒、传染性支气管炎病毒、传染性喉气管炎病毒以及减蛋综合症病毒,ＲＴ－ＰＣＲ扩增结果都呈阴性。禽流感病毒 Ａ／Ｇｏｏｓｅ／Ｇｕａｎｇｄｏｎｇ（Ｈ５Ｎ１）和 Ａ／Ａｆｒｉｃａｎ　Ｓｔａｒｌｉｎｇ／Ｅｎｇｌａｎｄ（Ｈ７Ｎ１）实验感染鸡样品 ＲＴ－ＰＣＲ检测与鸡胚病毒分离阳性率分别为３４／４２、３２／４２; ２４／５５、２４／５５, 二者符合率大于９５％。 ＲＴ－ＰＣＲ最少可检测到１０ｐｇ的病毒核酸。对山东某地发病鸡场样品进行ＲＴ－ＰＣＲ检测,只用６个小时就可得出准确的诊断结果,证明ＲＴ－ＰＣＲ检测方法敏感特异,可用于禽流感的快速诊断。     

Detection of Ehrlichia canis by polymerase chain reaction in dogs from Portugal

Antibodies against Ehrlichia canis, the cause of canine monocytic ehrlichiosis, have been reported previously in clinically ill and stray dogs from Portugal. In this study, the 16S rRNA gene of E. canis was detected by the polymerase chain reaction (PCR) in 12/55 (22%) of dogs with suspected tick-borne disease in the Algarve region in Portugal.     

用PCR技术从同一兔出血症病料扩增和检出2种病毒核酸

应用 Ｐ Ｃ Ｒ 和 Ｒ Ｔ Ｐ Ｃ Ｒ 技术,对兔出血症病毒核酸作了鉴定。分别设计合成 ２ 对 Ｐ Ｃ Ｒ 扩增特异性引物,即按兔出血症病毒中国分离的 Ｎ Ｊ株核酸序列合成 １ 对引物（ Ｎ Ｊ引物）,按德国分离的 Ｆ Ｒ Ｇ 株核酸序列合成另 １ 对引物（ Ｆ Ｒ Ｇ 引物）,进行 Ｐ Ｃ Ｒ 和 Ｒ Ｔ Ｐ Ｃ Ｒ。从纯化的病毒核酸样品和感染 Ｄ Ｊ Ｒ Ｋ 细胞的病毒核酸样品中,均扩增出 ２ 对引物范围内的特异性核酸片段, Ｎ Ｊ引物扩增产物为 ３６４ ｂｐ, Ｆ Ｒ Ｇ 引物扩增产物为 ５１３ｂｐ。模板用 Ｒ Ｎａｓｅ 消化后,仍出现 ３６４ ｂｐ 带,用 Ｄ Ｎａｓｅ Ｓ１ 消化,则此带消失;反之,用 Ｄ Ｎａｓｅ Ｓ１ 消化,出现５１３ ｂｐ 带,用 Ｒ Ｎａｓｅ 消化,则此带消失,证实前者模板为 Ｄ Ｎ Ａ,后者为 Ｒ Ｎ Ａ。 Ｓｏｕｔｈｅｒｎ 杂交试验证实, Ｐ Ｃ Ｒ扩增出的核酸片段与各自的地高辛标记特异性探针发生强阳性杂交反应。由此认为,在兔出血症病毒感染中,同时存在着 ２ 种不同核酸型的病毒。     

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID₅₀ tissue culture infective dose, 50%