生物素标记GFV－cDNA探针的制备及在检测葡萄扇叶病毒上的应用

以整合到质粒中的ＧＦＶ－ｃＤＮＡ为模板经ＰＣＲ合成了生物素标记的ＧＦＶ单、双链探针。用合成的探针对提纯的ｃＦＶ－ＲＮＡ＿２、感染ＣＦＶ的昆诺藜叶及１８株而萄进行ＤＮＡ－ＲＮＡ杂交检测表明：检测提纯病毒ＲＮＡ＿２的灵敏度为１．５ｐｇ／斑点,感染ＧＦＶ的昆诺藜提取液最高稀释度可达４０９６０倍;１１株显示典型扇叶症状的样品杂交结果均为阳性,且汁液稀释４００～８００倍仍能测出,７株不显示典型扇叶症状的葡萄中３株受到ＧＦＶ的侵染。单、双链探针最适使用浓度分别为１／２００及１／１００。     

生物素标记GFV—cDNA探针的制备及在检测葡萄扇叶病毒上的应用

以整合到质粒中的ＧＦＶ－ｃＤＮＡ为模板经ＰＣＲ合成了生物素标记的ＧＦＶ单、双链探针。用合成的探针对提纯的ＧＦＶ－ＲＮＡ２、感染ＧＦＶ的昆诺藜叶及１８株葡萄进行ＤＮＡ－ＲＮＡ杂交检测表明：检测提纯病毒ＲＮＡ２的灵敏度为１．５ｐｇ／斑点,感染ＣＦＶ的昆诺提取液最高稀释度可达４０９６０倍;１１株显示典型扇叶症状的样品均为阳性,且汁液稀４００～８００倍仍能测出,７株不显示典型扇叶症状的葡萄中３株受到ＧＦＶ     

生物素标记贾第虫全基因组DNA探针的制备及其特异性敏感性测定

用生物素标记的贾第虫全基因组ＤＮＡ探针,在斑点杂交试验中显示高度的敏感性和特异性。用它可检出１０ｎｇ贾第虫ＤＡ,１０＾３个贾第虫滋养体或包囊,且不与阴道毛滴虫、溶组织内阿米巴、弓形虫和ＢＡＢＬ／ｃ小鼠肝细胞ＤＮＡ,以及贾第虫患者粪便上清液发生交叉反应。本探针可用于贾第虫病病原体检测和虫株鉴定研究。     

生物素标记钙调素用于植物钙调素结合蛋白的检测

用生物素标记了花椰菜ＣａＭ。生物素标记的ＣａＭ具有与天然ＣａＭ相似的Ｃａ＾２＋依赖电泳特性,可激活ＣａＭ依赖性磷酸二酯酶,能够检测出５０ｎｇ的磷酸二酯酶。利用它建立了检测植物ＣａＭ结合蛋白的生物素－覆盖法并证实酶标亲和素可与胡萝卜愈伤组织内６４ｋＤ蛋白质非特异结合,因此将此法运用于植物材料时必要设置酶标亲和素处理的对照。用生物素－覆盖法检测胡萝卜愈伤组织形态过程中的ＣａＭ结合蛋白时可检出２,４－Ｄ     

丙型肝炎病毒E2基因DNA免疫实验动物研究

将编码丙型肝炎病毒（ＨＣＶ）Ｅ２蛋白４１７～７５０位氨基酸的ＤＮＡ片段　克隆到真核表达载体ｐｃＤＮＡ　３．１（－）中的ＣＭＶ　ＩＥ启动子下游,构建成ＨＣＶ　Ｅ２重组真核表达质粒　ｐｃＥ２。ＥＬＩＳＡ法检测ｐｃＥ２　ＤＮＡ免疫兔血清中的Ｅ２抗体变化和维持规律,结果显示免疫２０ｄ已有　抗体产生,３０ｄ后开始进入高峰,４０ｄ时达到最高值,至第９０ｄ抗体水平保持平稳,抗体滴度　达到１∶１６００左右。流式细胞计数仪（ＦＡＣＳ）检测ｐｃＥ２　ＤＮＡ免疫鼠ＣＤ４＋、ＣＤ８＋Ｔ淋巴细胞变　化情况,与注射空载体ｐＣＤＮＡ３．１（－）的阴性鼠相比,ＣＤ４＋淋巴细胞水平略有上升,ＣＤ８＋　细胞水平有较大升高,增幅达３５．４６％。免疫组化检测结果显示注射ｐｃＥ２的小鼠组织中有明显　的阳性着色,而注射ｐｃＤＮＡ３．１（－）的对照组小鼠免疫组化结果为阴性。以上结果表明：ｐｃＥ２　在实验动物内表达出的ＨＣＶ　Ｅ２蛋白可以引起免疫动物的体液免疫应答和细胞免疫应答,尤其　是ＭＨＣ－１限制性杀伤性ＣＤ８＋Ｔ淋巴细胞水平的提高对清除　病毒是十分有利的,因此ＨＣＶ　Ｅ２　ＤＮＡ免疫有可能成为预防和治疗ＨＣＶ感染的一条新途径。     

牛病毒性腹泻病毒基因组cDNA文库的构建

以牛病毒性腹泻病毒（ＢＶＤＶ）┥ｎＬ株的基因组ＲＮＡ为模板,经逆向转录酶作用,合成第一链ｃＤＮＡ,再以ＲＮａｄｓｅ／Ｈ与ＤＮＡ聚合酶Ｉ联合作用合成ｄｓｃＤＮＡ,并以ｄＣ同聚物尾化。ｐＵＣ８ＤＮＡ在Ｐｓｔ Ｉ酶解后,以ｄＧ同聚物尾化,两者退火构成重组质粒,转化到Ｅ．ｃｏｌｉ ＪＭ１０１受体菌中,另以γ－＾３２Ｐ－ＡＴＰ标记ＢＶＤＶ ＲＮＡ制备探针,通过菌落原位杂交筛选重组子。酶切分配表明重组质粒插入     

PCR制备地高辛素标记的探针检测轮伏病毒核酸

用聚合酶链反应（ＰＣＲ）技术,制备了轮状病毒第９基因部分片段的地高辛标记的ｃＤＮＡ探针。ＤＮＡ－ＲＮＡ斑点杂交表明该探针具有轮状病毒Ａ组的特异性,可检出１０ｐｇＨＲＶ－ＲＮＡ。实验中选择了粪便标本提取核酸后点膜和粪便上清直接点膜进行斑点杂交,两种方法的结果一致;、同时将斑点杂交法与ＰＡＧＥ法的结果进行了比较。实验结果表明用ＰＣＲ技术直接制备地高辛素标记的ｃＤＮＡ探针具有方便、快速、标记率高、特异性     

PCR制备地高辛素标记的探针检测轮状病毒核酸

用聚合酶链反应（ＰＣＲ）技术,制备了轮状病毒第９基因部分片段的地高辛标记的ｃＤＮＡ探针。ＤＮＡ－ＲＮＡ斑点杂交表明该探针具有轮状病毒Ａ组的特异性,可检出１０ｐｇＨＲＶ－ＲＮＡ。实验中选择了粪便标本提取核酸后点膜和粪便上清直接点膜进行斑点杂交,两种方法的结果一致;同时将斑点杂交法与ＰＡＧＥ法的结果进行了比较。实验结果表明用ＰＣＲ技术直接制备地高辛索标记的ｃＤＮＡ探针具有方便、快速、标记率高、特异性强的特点,具有一定的应用前景。     

应用生物素标记DNA探针检测伪狂犬病病毒的研究

应用生物素标记ＤＮＡ探针检测伪狂犬病病毒的研究王琴,郭万柱,阴文奇（四川农业大学动物科技学院,四川雅安,６２５５０１４）关键词伪狂犬病毒,核酸,杂交,检测核酸杂交技术已广泛用于检测畜禽疱疹病毒和其它动物病毒,已报道ＰＲＶ－ＤＮＡ的探针方法有ＲＮＡ－Ｄ...     

应用DIG标记探针杂交检测IBDV

本试验用地高辛（Ｄｉｇｏｘｉｇｅｎｉｎ,ＤＩＧ）标记的探针建立了核酸杂交检测传染性法氏囊病病毒（ＩＢ－ＤＶ）的方法,并在敏感性方面同琼脂扩散试验进行了比较。探针来源于ＩＢＤＶＳＴＣ－ｌｌ９和ＳＴＣ－２４３ｃＤＮＡ。杂交方法的敏感性试验表明,核酸杂交可以检测到０．１ｐｇ的ＩＢＤＶＲＮＡ;与多个毒株核酸的杂交则显示出标记的探针可以作为通用试剂用于ＩＢＤＶ早期感染的诊断;而同琼脂扩散试验的比较则说明,杂交方法比常规的免疫沉淀反应敏感１０４倍以上。除此之外,由于杂交方法特异以及非放射性探针操作方便,因而具有很大的应用前景。     

利用RT—PCR扩增和分析柑桔裂皮病类病毒

参考国外CEVd-A株中央保守区段(C区)和左端区段(T区),设计并合成引物Cl(-)、C2( )、C3( )、T1(-)、T2( )。对感染 CEVd中国分离物的柑桔(Citrus L.)和香橼(Citrus medica L.)总核酸进行了cDNA第一链合成和PCR扩增,其中C1/C3、C1/C2分别能从感病香橼和柑桔总核酸中扩增出210bp和370bp左右的特异DNA,分别相当于CEVd的左半部和全长片段,T1/T2未能扩增出产物;健康香橼和柑桔总核酸中均未能扩增出产物。扩增结果用DIG标记的CEVd-cDNA探针进行了确证。扩增结果说明:CEVd中国分离物在左端T区与CEVd-A株存在差异。PAGE-银染法分析扩增产物表明:建立的RT-PCR方法可从约0.1ng柑桔总核酸中扩增出全长CEVd-cDNA。     

Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin.

A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100-400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 X 10(-18) mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32p-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.     

应用聚合酶链式反应扩增和光敏生物素标记的cDNA探针检测马铃薯纺锤块茎类病毒

以含马铃薯纺锤块茎类病毒(potato spindle tuber viroid,PSTV)RNA的总核酸为模板,加入人工合成的互补DNA引物,用反转录酶合成PSTV cDNA;在聚合酶链式反应系统中,用两个PSTV特异性引物进行cDNA扩增,用以制备光敏生物素标记的PSTV cDNA探针。用此探针进行斑点杂交检测含PSTV的马铃薯核酸提取液和汁液均出现阳性杂交信号,而健康马铃薯的核酸提取液和汁液的结果均为阴性。光敏生物素标记探针检测纯化PSTV的灵敏度可达5pg;检测感染PSTV的马铃薯块茎汁液的可测出最高稀释度为1:400。     

Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes.

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.     

A Comparison of Digoxigenin and Biotin Labelled DNA and RNA Probes for in Situ Hybridization

A number of in situ hybridization protocols using digoxigenin or biotin labelled probes were assessed for viral nucleic acid detection in formalin fixed, paraffin embedded tissue. Single-step detection protocols for biotin labelled probes produced low sensitivity; however, enzyme based one-step detection protocols for digoxigenin probes produced high sensitivity for both RNA and DNA systems. For both probe types, multistep detection protocols produced equally high sensitivity. Use of an enhanced APAAP procedure for digoxigenin labelled probes acheived maximal sensitivity without use of biotin-streptavidin reactions. The sensitivity of nucleic acid detection obtained with a digoxigenin labelled probe is comparable to that obtained using biotin. Digoxigenin labelled probes for nucleic acid detection are recommended for tissues with endogenous biotin.     

Novel biotin linker with alkyne and amino groups for chemical labelling of a target protein of a bioactive small molecule

We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.     

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of ³²P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed.These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques.The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.     

An alternative form of IL-18 in human blood plasma: complex formation with IgM defined by monoclonal antibodies

Monoclonal Abs 21 and 132 were raised against human functionally inactive rIL-18, and plasma IL-18 levels were determined by the sandwich ELISA established with these mABS: Plasma IL-18, designated type 2, was detected by this ELISA, and the levels found were not consistent with those obtained with the commercially available kit for determination of functionally active IL-18 (type 1). Type 1 was detected in all volunteers, whereas type 2 was detected in approximately 30% of healthy subjects, and the levels of type 2 in their blood plasma were high (25-100 ng/ml) compared with those of type 1 (0.05-0.3 ng/ml). We purified IL-18 type 2 from blood plasma of volunteers with high IL-18 type 2 concentrations, and its M(r) was determined to be 800 kDa by SDS-PAGE and molecular sieve HPLC. The purified 800-kDa protein, either caspase-1-treated or untreated, expressed no or marginal IL-18 function in terms of potentiation of NK-mediated cytolysis and IFN-gamma induction, and it barely bound IL-18R-positive cells. N-terminal amino acid analysis indicated that the purified protein was IgM containing a minimal amount of IL-18 proform and its fragment. Again, the purified IgM from IL-18 type2-positive volunteers exhibited cross-reaction with mAb 21 against IL-18. This band was not detected with 125-2H, an mAb against functionally active IL-18. Hence, human IgM carries functionally inactive IL-18 forming a disulfide-bridged complex, and this IL-18 moiety is from 10- to 100-fold higher than the conventional type 1 IL-18 in blood circulation in approximately 30% normal subjects.     

Rapid detection and identification of biological and chemical agents by immunoassay, gene probe assay and enzyme inhibition using a silicon-based biosensor

A rapid biosensor assay procedure that utilizes biotin streptavidin mediated filtration capture onto nitrocellulose membrane, in conjunction with a silicon-based light-addressable potentiometric sensor (LAPS) was developed for detection and identification of biological and chemical threat agents. Sandwich immunoassays, nucleic acid hybridization assays and enzyme inhibition assays are described. For immunoassays, the lower limits of detection (LOD) per 100-microl sample were approximately 5 pg/ml for protein (Staphylococcal enterotoxin B), 2 ng/ml for virus (Newcastle disease virus), and 20 ng/ml for vegetative bacteria (Brucella melitensis). In a dual gene probe assay format, the LOD was 0.30 fmol (1.8 x 10(8) copies per 60-microl) of single stranded target DNA. Enzyme inhibition assays on the LAPS using acetylcholinesterase were able to detect soman and sarin in aqueous samples at 2 and 8 pg (100 and 600 pM), respectively. The assays were easy to perform and required a total time equal to the reaction period plus about 15 min for filtering, washing and sensing. The assay format is suitable for detection of a wide range of infectious and toxic substances. New assays can be developed and optimized readily, often within 1 or 2 days.     

双链探针实时荧光PCR核酸检测新技术研究*

目的:采用一种“双链探针”实时荧光PCR技术,提高HBV核酸检测灵敏度,并在同一反应管中实现代谢酶CYP2C19^*2基因型检测。方法:采用双链探针与TaqMan探针同时检测不同浓度HBV血清样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和结果统计分析;采用双链探针检测代谢酶CYP2C19^*2不同基因型样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和基因型确定。结果:不同浓度HBV血清样本检测,双链探针荧光本底低,检测灵敏度更高,与TaqMan探针检测结果相比,两者核酸检测Ct值存在显著性差异(P<0.05);双链探针检测36份样本的代谢酶CYP2C19^*2基因型,检测结果与Sanger测序结果完全一致。结论:双链探针实时荧光PCR检测技术可完成目的基因的高灵敏核酸检测,也可实现基因型分析。