应用RT—PCR方法检测鸭病毒性肝炎新型和I型病毒

鸭病毒性肝炎病毒是危害养鸭业的重要病毒性传染病,主要感染低日龄雏鸭,临床上分离到的病原主要为鸭病毒性肝炎I型病毒和鸭病毒性肝炎新型病毒,为了加快本病的检测速度,尽快确诊病原,根据GeneBank上两种毒株的基因组序列,选择两种毒株的差异基因序列分别设计了一对特异性的检测引物,将两对引物放在一个PCR反应体系中,两种毒株分别进行特异性扩增,根据扩增片段的大小进行鉴别,建立了可以同时检测鸭病毒性肝炎病毒I型和新型毒株的复合RT—PCR方法,该检测方法快速,特异性强,灵敏性高,可以用于鸭病毒性肝炎病毒的分型鉴别检测。     

李氏杆菌研究进展

李氏杆菌病是一种重要的人畜共患传染病 ,主要由单核细胞增多性李氏杆菌引起 ,呈世界性分布。文章回顾了李氏杆菌的分离鉴定、培养、分型等生物学特性以及与其毒力有关的基因功能方面的研究进展 ,着重就该病诊断和检测的重要性以及 EL ISA和 PCR检测方法进行了概述 ,同时对该病的免疫预防和今后的研究重点进了探讨     

HRP直接标记属特异性基因探针检测沙门氏菌的研究

以活化辣根过氧化物酶复合物（ＨＲＰ—ＰＢＱ—ＰＥＩ＋—ＮＨ３＋）直接标记沙门氏菌属特异性ＤＮＡ探针ｐＬＳ２和ｐＬＳ３，探针与靶ＤＮＡ杂交后催化发光底物，经增强型化学发光反应（ＥＣＬ），用普通Ｘ光胶片自显影（ＣＰＤ）检测沙门氏菌。经狭缝杂交（Ｓｌｏｔｂｌｏｔ），该法标记探针均可检测到０．１ｐｇ的纯质粒ＤＮＡ及１０３个未经培养的鼠伤寒沙门氏菌。Ｄｏｔ—ｂｌｏｔ杂交结果证明，ＨＲＰ标记的探针仅与沙门氏菌属细菌杂交，而与试验的其他肠道非沙门氏菌不杂交。本研究表明，ＨＲＰ直接标记基因探针化学发光自显影法检测沙门氏菌，安全、快速、简便，且有高度的敏感性和特异性，是一种有较大应用前景的非放射性标记探针杂交检测方法。     

用套式PCR方法对几种猪用冻干疫苗的PCV2检测

参照Genbank已发表的猪圆环病毒2型的基因序列,取其比较保守的基因片段ORF2,利用引物设计软件Oli-go5.0设计两对PCV2型特异的引物,其中第一对引物扩增跨度为ORF2全基因片段,长度为702 bp,第二对引物扩增ORF2中间的一个小片段,跨度大小为494 bp.首先用第一对引物扩增阳性DNA,阳性DNA的浓度从10-1稀释到10-11,然后以第一次PCR产物为模板,用第二对引物进行PCR,发现这种套式PCR的方法比用普通PCR灵敏度提高102倍.同这种套式PCR方法对广东省市场上出售的几种猪用弱毒疫苗进行猪圆环病毒2型检测,结果发现这几种疫苗全部为阴性,本实验说明目前广东市场上出售的弱毒疫苗在制作过程中没有受到PCV2的污染,解除了广大猪场主的顾虑.     

链球菌Gapc基因检测及其序列分析

根据已发表的猪链球菌2型3-磷酸甘油醛脱氢酶(Gapc)基因保守区域设计并合成1对特异性引物,对10株不同来源链球菌Gapc基因进行PCR扩增,并选择PCR产物进行克隆并测序分析,结果在10株不同来源链球菌菌株中有8株可扩增出与预期大小(1011 bp)相一致的DNA片段;选取4株细菌PCR产物进行克隆测序,发现其片段大小为1009或1012 bp,与GenBank参考菌株Gapc基因序列的核苷酸同源性为85.3%~98.3%,氨基酸同源性为87.2%~99.4%;生物学软件分析结果显示Gapc基因在链球菌属细菌中相对保守,且具有较多的潜在抗原表位。本研究为链球菌检测技术和核酸疫苗的研究奠定了基础。     

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。     

百色市高致病性猪繁殖与呼吸综合征病毒抗体检测结果分析

为评估百色市猪群高致病性猪繁殖与呼吸综合征（HP-PRRS）免疫效果,2012年—2013年在66个规模养猪场和249个农村散养户分别采集猪血清样品465份和644份,使用间接 ELISA 检测 HP-PRRSV 抗体,结果显示,现2012年 HP-PRRSV 抗体平均阳性率为68．02％,2013年 HP-PRRSV 抗体平均阳性率为75．32％;HP-PRRSV 一免抗体平均阳性率为63．91％,二免抗体平均阳性率为83．60％;HP-PRRS 灭活苗免疫抗体平均阳性率为75．42％,HP-PRRS 弱毒苗免疫抗体平均阳性率为70．86％。说明2013年 HP-PRRSV 抗体平均阳性率高于2012年,二免的抗体平均阳性率高于一免;HP-PRRS 灭活疫苗的初步使用效果良好。     

Evaluation of a Bacillus stearothermophilus tube test as a screening tool for anticoccidial residues in poultry

A Bacillus stearothermophilus var. calidolactis C953 tube test was evaluated for its ability in detecting the residue of selected anticoccidial drugs in poultry, specically sulfamethazine, furazolidone, and amprolium. Various concentrations of each drug were injected into chicken liver and kidney tissues and these tissues were tested to determine the drug detection limits for each drug. The detection limit was defined as the drug concentration at which 95% of the test results were interpreted as positive. The limits of detection in liver tissue were 0.35 µg/ml for furazolidone, 0.70 µg/ml for sulfamethazine and 7.80 µg/ml for amprolium. In kidney tissues, they were 0.30 µg/ml for furazolidone, 0.54 µg/ml for sulfamethazine, and 7.6 µg/ml for amprolium. It was concluded that this tube test could be used to screen for the residue of these three drugs in poultry.     

Syndromic surveillance using laboratory test requests: A practical guide informed by experience with two systems

Syndromic surveillance systems can enhance early disease warning, endemic disease monitoring, or help to accumulate proof of disease freedom. In order to provide immediate feedback to achieve these goals, the health data sources scanned should be acquired continuously, in an automated fashion, and should be stored electronically. Recognizing that data from diagnostic test requests often meet these requirements, two systems designed to automatically extract surveillance information from animal laboratory databases have been developed and are described in this paper. These systems are designed to contribute to early disease detection, as well as the timely management of epidemiological information, in a province of Canada and in Sweden, the areas served by the diagnostic laboratories concerned. Classifying in-coming requests into syndromes, the first step, was the most time-consuming and the least portable step between the two systems. The remaining steps were more easily adjusted from one system to implementation in the other. These steps included: retrospective evaluation of data to create baseline profiles following the removal of excessive noise and aberrations; the identification of temporal effects; prospective evaluation of detection algorithms; and finally real-time monitoring and implementation. Building upon the institutions’ existing data management software, all steps to use those data for the purposes of syndromic surveillance were set up using open source software; as a result this approach could be readily adopted by other institutions. Relatively straight-forward development and maintenance is expected to lead to the incorporation of these systems into each institution's surveillance processes, becoming an indispensable tool for diagnosticians and epidemiologists, as well as stimulating further technical development of such systems.     

猪附红细胞体PCR-微孔板杂交-酶免疫检测方法的研究

以猪附红细胞体和多种血营养菌16S rRNA基因的保守区设计合成引物HM-f/HM-r,在反向引物的5′端用生物素标记,以猪附红细胞体广东株16S rRNA基因的可变区序列设计种特异性寡核苷酸探针,探针的5′端用地高辛标记,成功地建立了猪附红细胞体PCR-微孔板杂交-酶免疫检测方法。通过测定不同浓度PCR产物对应的微孔板杂交-酶反应显色OD值,用SPSS统计软件进行回归分析,得到回归方程为y=2.1012-0.4492×logx,其相关系数R为0.977,显示对未知样品可进行定量检测。该方法与猪的多种细菌性病原、猫血巴尔通氏体CA株和E.wenyonii无交叉反应,其敏感性比常规PCR琼脂糖电泳检测提高了近100倍。用建立的方法对38份临床样品进行检测,结果有12份检测结果为阳性。