Taxonomy of pasteurella anatipestifer. 1. DNA base composition and DNA-DNA hybridization analysis

DNA was isolated from 15 strains of Pasteurella anatipestifer and from one strain each of Moraxella nonliquefaciens, M. bovis, Pasteurella multocida, P. haemolytica, P. gallinarum, P. pneumotropica, and P. ureae. The guanine-plus-cytosine contents of P. anatipestifer ranged from 32 to 35 mole %, whereas those of Moraxella and Pasteurella spp. were much higher, ranging from 40 to 45 mole %. DNA-DNA hybridization analysis revealed that homology of nine P. anatipestifer strains to strains ATCC 11845 and PA 15 was 52 to 100%, whereas homology of Moraxella and Pasteurella strains to these strains was only 3 to 17%. Similarly, homology of P. anatipestifer strains, Moraxella, and Pasteurella species other than P. multocida to P. multocida reference strain P-2192 was low. These results strongly suggest that P. anatipestifer is genetically unrelated to either Pasteurella or Moraxella.     

鸭疫里默氏杆菌菌体蛋白双向电泳技术的建立

为了建立鸭疫里默氏杆菌菌体蛋白的双向电泳技术,获得分辨度高、重复性好的双向电泳图谱。利用适当的裂解液处理鸭疫里默氏杆菌,提取全菌蛋白;采用pH值4～7,24cm干胶条,0.8 mg菌体蛋白进行双向电泳;硝酸银染色后获得的双向电泳图谱,并利用I mage MasterTM2D Platinum5.0图象分析软件进行分析,所得的数据用SPSS 15.0软件进行统计分析。结果得到了(800±26)个蛋白斑点,蛋白主要集中在pI值4.13～7.40之间,重复胶的匹配点数为(600±20),匹配率为75.2%。建立了鸭疫里默氏杆菌菌体蛋白双向电泳技术,2-DE图谱中蛋白位点的分辨率和重复性比较高,为进一步研究其蛋白质组学奠定了基础。     

Thin layer chromatography convulsant screen extended by gas chromatography-mass spectrometry.

Acute onset convulsive disorders in the canine may result from exposure to a variety of toxicants including strychnine, insecticides, metaldehyde, zinc phosphide, methylxanthines, drugs of abuse, bromethalin, and the tremorgenic mycotoxins (roquefortine and penitrem A). Although several of the above can be identified in a single gas chromatography-mass spectrometry (GC-MS) screen most have to be determined by separate tests. This report describes a modification of the strychnine extraction procedure, which allows thin layer chromatographic (TLC) identification of strychnine, bromethalin, roquefortine, and penitrem A in suspect baits, stomach contents or vomitus, and extends the identification to a wide variety of drugs, pesticides, and environmental contaminants by GC-MS. Samples were mixed with base, extracted into CH2Cl2 and the organic fraction back-extracted with acid. The organic fraction (neutrals) was purified by gel permeation chromatography (GPC) and analyzed by TLC to determine penitrem A and bromethalin. The acidic aqueous fraction was adjusted to pH > 9 and extracted into CH2Cl2. The resulting CH2Cl2 layer (bases) was then analyzed by TLC to determine strychnine and roquefortine. The organic basic and neutral fractions were recombined with a late eluting GPC fraction and analyzed by GC-MS. Of 312 samples analyzed by TLC from 1995 to 2001, 35 were positive for strychnine alone, 58 were positive for both roquefortine and penitrem A, 4 were positive for roquefortine alone, and 1 was positive for bromethalin. None of the samples were positive for penitrem A alone. Samples negative by TLC were analyzed by the GC-MS extended procedure since mid-1999, and 14 have shown positive for a wide variety of compounds with convulsant activity.     

Molecular fingerprinting of Riemerella anatipestifer by repetitive sequence PCR.

Riemerella anatipestifer is a gram-negative rod-shaped bacterium associated with epizootic infections in poultry. A total of 35 R. anatipestifer isolates including the type strain ATCC11845T, reference and field strains for 18 different serotypes were characterized by repetitive sequence based-PCR (rep-PCR) with outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence. This technique was applied by using either extracted genomic DNA or preparation of whole bacterial cells harvested directly from plate cultures. Rep-PCR discriminated the R. anatipestifer isolates into 19 electrophoretic types. DNA fingerprints obtained from rep-PCR of extracted genomic DNA or from preparations of whole cells yielded comparable patterns. Substantial variation was seen among the rep-PCR fingerprints of different serotypes. Moreover, different polymorphisms of the rep-PCR fingerprints were evident among epidemiologically unrelated isolates of the same serotype. These results suggest the presence of repetitive extragenic palindromic-like elements within the genome of R. anatipestifer that can be used in some isolates to discriminate between different strains belonging to the same serotype. Rep-PCR may serve as a useful molecular tool for subtyping R. anatipestifer isolates for epidemiologic investigations. The whole cell procedure offers the advantage of ease of performance requiring only small quantities of cells.     

Determination of chlorinated pesticide residues by gas chromatography]

Possibilities were tested of using several phases of chromatography used in toxicological laboratories (3% OV-1, 3% OV-17, 3% NPGS + 0.75% TA) and mixed fillings (3% OV-17, 7.5% QF-1, 3% XE-60 in a 2 : 2. : 1 ratio) in the separation of chlorinated pesticides. In the tested fillings, the retention volumes and Kováts indices were measured for 16 chlorinated hydrocarbons. The amounts of chlorinated hydrocarbon residues in water and eggs were also determined.     

气相色谱法测定骆驼奶中的胆固醇

胆固醇的分析方法主要有比色法、薄层法和酶催化法，在应用于食品的测定中，它们具有操作繁琐、选择性低等缺点，而高效液相色谱法用于分析胆固醇时，除了麦角固醇等具有共扼体系的一部分化合物之外。在紫外区都不具有最大吸收，因而没有可供使用的高灵敏度检测器，此外，它对胆固醇和豆固醇分离效果差。为此，笔者等采用气相色谱法对骆驼奶中的胆固醇进行了测定。     

应用PCR快速检测鸭疫里默氏杆菌的研究

根据鸭疫里默氏杆菌（RA）16SrDNA基因序列设计引物,对1株RA阳性株、9株临床分离鉴定的RA、3株大肠杆菌、1株多杀性巴氏杆菌和1株沙门氏菌进行PCR扩增,结果所有RA均出现643bp特异性扩增条带,其余非RA则均未扩增出特异性条带,表明该对引物及建立的PCR具有很强的特异性。优化的PCR反应体系能检出RA的最低DNA量为20pg。菌落直接PCR是增强RA快速检测鉴定的手段。应用PCR对病料组织直接进行RA检测,脑组织为首选检测对象,具有良好的实际应用意义。     

2型鸭疫里默氏杆菌的分离

从北京地区两个发病的鸭场中分离到两株细菌,经过染色,生化鉴定,凝集试验和琼扩试验,鉴定为２型鸭设里默氏杆菌,经过致病性试验,其中一株有很强烈致病性,另一株在实验室条件下,人工感染小鸭未能引起小鸭发病。     

气相色谱法测定猪肉脂肪酸组成

实验采用极性毛细管柱气相色谱法测定了猪肉脂肪酸组成的相对百分含量和实际含量。测定结果表明,此方法能准确分离出猪肉中的主要8种脂肪酸:豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸、亚麻酸和花生酸,有较好的精密度和重复性、可靠性。     

家兔血清中苦马豆素的气相色谱测定方法

建立家兔血清中苦马豆素(SW)浓度的气相色谱测定方法,为SW的毒理学和免疫学研究提供条件。饲喂家兔甘肃棘豆草粉后,分别于5、10、20d采集血液并分离血清;血清经过丙酮沉淀、超声和离心净化处理后,冷冻干燥;冻干物经硅烷化试剂衍生化反应,用气相色谱内标法测定血清中SW的含量。结果显示,测定SW的线性范围为0.00008～2.5g/L,相关系数γ=0.9996,检出限为0.00008g/L。在0.001、0.01、0.1、1.0g/L分别添加SW的回收率为89.81%～96.23%,相对标准偏差为2.46%～4.35%。结果表明,该方法准确、灵敏、快速、简便,适用于家兔血清中SW含量的测定。     

鸭疫里默菌环介导等温扩增检测方法的建立

采用对应于鸭疫里默菌16SrRNA基因442~461bp序列的5条特异引物,建立了环介导等温扩增(LAMP)反应体系,加入试剂盒提取的鸭疫里默菌DNA后,置63℃反应60min,通过监测反应液浊度判断结果。在此基础上,用煮沸10min的方法代替试剂盒提取DNA方法,并用显色方法代替浊度检测和琼脂糖凝胶电泳方法判断结果,通过敏感性、特异性、病原消长规律试验验证方法的准确性。另外,对临床采集的135只患病鸭的心血、脑组织、肝脏和心脏共540份样品提取DNA后,用LAMP和PCR进行检测,同时进行细菌分离和鉴定,比较各种方法检测结果的符合率。结果显示,菌液提取DNA后,LAMP和PCR方法最低分别可检测到2和10个CFU的鸭疫里默菌。菌液煮沸代替试剂盒提取DNA方法,使LAMP的敏感性降低5倍。显色法、浊度检测和琼脂糖凝胶电泳方法判断结果的敏感性和特异性相同。细菌分离法共获得201株鸭疫里默菌,且经LAMP与PCR法检测均呈阳性,LAMP与PCR法检出阳性样本总数分别为271和254份,LAMP方法阳性检出率高于PCR方法。建立的鸭疫里默菌LAMP检测方法具有较高的敏感性,进一步用菌液煮沸方法代替DNA提取法虽然使敏感性降低5倍,但节省了DNA提取步骤,再结合显色技术使反应结果直观可见,使其临床应用更加便捷。     

饲料中盐酸克伦特罗的气相色谱-质谱法测定分析

探索了饲料中盐酸克伦特罗的气相色谱-质谱法测定方法。饲料试样中盐酸克伦特罗经0.01mol/l盐酸溶液提取,乙醚脱脂净化,乙酸乙酯提取后蒸发至干,用乙醇溶解后加样到氧化铝柱上,用0.01mol/l盐酸溶液洗脱,蒸发至干后,分别用三甲基氯硅烷(TMCS)和乙酸酐衍生,采用GC/MS/SIM方式进行测定,均达到了保留时间之差不大于2s(分别为0.002min,0.004min),匹配度大于800(分别为863,923)的定性要求,符合农业部行业标准NY/T468—2001。     

气质联用检测饲料中氯霉素的方法研究

用乙腈／4％氯化钠（1：1）混合液提取饲料中氯霉素,采用两种方法对提取物进行净化分离。一种方法用Oasis HLB固相萃取柱代替传统的C18柱对提取物进行净化富集,另一种方法不使用固相萃取柱。净化后的提取物用BSTFA＋TMCS进行衍生,气相色谱-离子肼质谱联用仪检测,采用SIM模式电离,选择离子为225、208、178,通过对净化、提取及质谱条件的优化,该方法对不同饲料样品中氯霉素的加标回收率在76.1％-102.3％,相对标准偏差小于10％,方法的检出限为0.1mg/g。     

气相色谱/质谱法测定饲料中三唑仑的含量

本实验建立了检测饲料中三唑仑的气相色谱/质谱法。样品经有机溶剂提取,阳离子固相萃取小柱净化后注入气相色谱质谱仪进行测定,并且经质谱确证。结果表明,三唑仑在0.1～5.0μg/mL范围内具有良好的线性关系,方法的回收率、精密度较高。     

毛细管气相色谱法测定发酵液中丙酸的含量

丙酸(propionicacid,以下简称PA)是一种理想而又有前途的粮食、食品和饲料防腐剂,在国外饲料工业中的应用最为广泛犤1,2犦。我国作为世界饲料生产大国,对丙酸的需求量很大,并且呈现逐年递增的态势。据统计,仅1999年,我国就从国外进口丙酸9880t犤2犦。预计到2003年,我国的丙酸需求量将超过10000t,而我国丙酸的年产量却不足200t犤2犦。目前,国际上主要采用化学合成法生产PA,也有学者开始研究利用微生物(如丙酸杆菌)发酵法生产PA犤3犦。微生物发酵法的原料来自农副产品,能充分利用可再生资源,符合绿色化工发展方向,因而日益受到人们的重视。…