Canine Haematopoietic Chimerism Analyses by Semiquantitative Fluorescence Detection of Variable Number of Tandem Repeat Polymorphism

Canine models are successfully applied to the study of haematopoietic stem cell transplantation (HSCT). Monitoring of haematopoietic donor/recipient chimerism is of major significance in detecting and quantifying engraftment or graft rejection of the donor-derived haematopoietic cells after transplantation. Radioactive analyses of polymorphic microsatellite markers are commonly used for chimerism analyses. We describe an improved, non-isotopic method that is based on the analysis of microsatellite markers in donor and recipient cells using capillary electrophoresis and fluorescence detection. Artificial mixtures of donor and recipient DNA that were generated from peripheral blood mononuclear cells from dog leukocyte antigen-identical siblings were used to analyse the sensitivity of the assay. DNA from dogs that had received HSCT were also analysed in order to demonstrate the feasibility of the method in vivo. For chimerism analyses, six different microsatellite loci were systematically amplified using fluorescent PCR primer. The fluorescent polymerase chain reaction products were separated by capillary electrophoresis using POP4 on a 310 ABI Prism Genetic Analyzer. After electrophoresis, fluorescence signals were automatically sized and quantified using GeneScan software. The method described provides an accurate assessment of haematopoietic chimerism in the canine model with significantly reduced hands-on time compared to conventional gel electrophoresis.     

圆点印迹法检测葡萄斑点病毒技术研究

采用圆点印迹法检测GFkV，结果表明：Tris-HCl和PBS在提取GFkV时效果相同，在带毒葡萄植株中韧皮部的GFkV含量较高。     

禽流感病毒夹心ELISA快速检测方法的研究

以禽流感病毒(AIV)湖北分离株(H9N2亚型)提纯物为免疫原，制备出鸡抗AIV和兔抗AIV抗体，经用琼脂扩散法测得效价分别为1:32和1:16。以纯化的鸡抗AIVIgG为包被抗体，兔抗AIVIgG为第二抗体，建立了检测AIV抗原的夹心ELISA法。结果表明，鸡抗AIVIgG的最佳包被浓度为1μg/mL，兔抗AIV IgG的最适工作浓度为5μg/mL；对已知的阳性样品，用夹心ELISA法测得的病毒滴度比血球凝集滴度高16倍以上，且能检出其它亚型的禽流感病毒；与新城疫病毒、传染性支气管炎病毒、减蛋综合征病毒、传染性喉气管炎病毒、传染性法氏囊病病毒、鸡痘病毒、马立克氏病病毒等无交叉反应，说明本方法有很高的特异性及敏感性。对14个鸡场送检的、患有呼吸道疾病或有腹膜炎、眼炎、产蛋下降、怀疑为禽流感感染的病鸡进行了检测，结果有7个鸡场为阳性。本方法的建立为禽流感病鸡群的临床检验提供了一种方便、敏感、快速的检测方法。     

应用多重聚合酶链式反应检测鸡毒支原体、鸡滑液囊支原体和禽衣阿华支原体的研究

根据鸡毒支原体 (MG)、禽衣阿华支原体 (MI)、鸡滑液囊支原体 (MS)的基因文库 ,设计了 3对分别与MG、MI、MS某段基因序列互补的引物。用这 3对引物对同一样品中的MG、MI、MSDNA模板进行多重聚合酶链式反应 (PCR)扩增 ,结果均同时得到了 3条特异性的大小与实验设计相符的 732bp (MG)、 2 99bp (MI)、 2 0 7bp (MS)多重的PCR扩增带 ,而对其他 6种禽病病原的PCR扩增结果均为阴性 ;敏感性测定结果能同时检出 1pg的MG、MI和MSDNA模板     

用重组核衣壳蛋白ELISA检测PRRSV抗体的研究

ＰＲＲＳＶ重组Ｎ蛋白ＥＬＩＳＡ检测人工感染猪血清７５份,与ＩＤＥＸＸ公司ＥＬＩＳＡ试剂盒及ＩＦＡ的符合率分别为９７．３％和１００％。检测疫苗免疫猪血清,阳性率１００％。在免疫后６ｄ就能检出抗体,比ＩＦＡ敏感。检测８５份我国田间猪血清,阳性率１８．８％。     

禽流感病毒重组核蛋白ELISA诊断技术的研究

用表达禽流感病毒（ＡＩＶ）核蛋白基因的杆状病毒感染Ｓｆ９昆虫细胞。以其表达产物制备抗原,建立了以杆状病毒系统表达的ＡＩＶ核蛋白为抗原的禽流感间接酶联免疫吸附试验诊断技术（ｒＮＰ－ＥＬＩＳＡ）。其抗原最适包被量为０．６μｇ／孔,等检血清最适稀释度为１：２００,酶标抗体使用浓度为１：１０００。根据对１４０份ＳＰＦ鸡血清检测结果的统计分析,确定其判定标准为ＯＤ均为阴性;检测Ａ型ＡＩＶ１５个不同亚型（Ｈ１￣Ｈ１５）毒株的特异性血清均为阳性;对人工接种ＡＩＶ的ＳＰＦ鸡第３天即能检出抗体,到第１６２天试验结束时检测仍为阳性。其批内和批间重复试验的变异系数分别在２．９％￣７．２％和３．４％￣９．８％之间。对３１３８份鸡血清进行监测,ｒＮＰ－ＥＬＩＳＡ与全病毒间接ＥＬＩＳＡ与全病毒间接ＥＬＩＳＡ（ＡＩＶ－ＥＬＩＳＡ）、琼脂扩散     

乳胶凝集试验与血清中和试验检测猪伪狂犬病血清抗体的 …

应用血清中和试验（ＳＮＴ）和伪狂犬病乳胶凝集试验（ＬＡＴ）诊断试剂盒对两种伪狂犬病是性血清、伪狂犬病病毒（ＰＲＶ）高兔血甭及６０份被检猪血清进行了ＰＲＶ抗体效价测定和相关性分析,两种方法测得的抗体效价之间呈强相关性（ｒ＝０．９６）,且ＬＡＴ效价比ＳＮＴ一般高出一个滴度;能干为自３５个猪场的４１４份猪血清进行了ＰＲＶ抗体检测,并与ＳＮＴ检测结果进行了对比,结果在ＳＮＴ检测为阳笥的１７１份血清中,ＬＡ     

Detection of Tobamoviruses from Soils by Non-precoated Indirect ELISA

A method for detecting tobamoviruses from field soils was developed using non-precoated indirect enzyme-linked immunosorbent assay (Id-ELISA). Absorbance values in Id-ELISA were relatively low after directly applying Pepper mild mottle virus (PMMoV)-infested soil extract. However, heat treating the soil extract before application greatly enhanced the absorbance values. The heat treatment was essential for the Id-ELISA detection of tobamoviruses from infested soil, although the efficiency of virus recovery varied depending on the properties of soil. The number of local lesions in the infectivity assay was consistent with the absorbance values in Id-ELISA. Moreover, the absorbance values in Id-ELISA were correlated with the incidence of soil transmission of PMMoV. Thus, Id-ELISA combined with heat treatment is a practical technique for the diagnosis of infestation with Tobamovirus in field soils, Gray Lowland soil and Sand-dune Regosol. Received 4 October 1999/ Accepted in revised form 9 December 1999     

Molecular Variability of the Capsid Protein of the Prune Dwarf Virus

Sequences of the capsid protein gene and the preceding intergenic region of eleven isolates of prune dwarf virus from central Europe were determined. The isolates were obtained from plum, cherry and peach trees. Comparison of all sequenced isolates (including two sequences published previously) revealed high (88%) conservation of the capsid protein gene. The highest degree of identity was observed in the C-terminal half, where only 13 amino acid substitutions could be observed in contrast to the N-terminal half with 22 substitutions. No reasonable correlation between amino acid substitutions and host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other ilarviruses revealed apple mosaic virus, elm mottle virus, lilac ring mottle virus and prunus necrotic ringspot virus as the most related to prune dwarf virus. Unlike the isolates of related prunus necrotic ringspot virus all the isolates of prune dwarf virus shared extensive conservation of the intergenic region. Portions of RNA3 were selected for design of universal primers for PCR detection.