快速ELISA诊断猪旋毛虫病的研究

在快速间接ＥＬＩＳＡ中将被检血清样品和酶结合物一起保温，即简化了操作步骤，又比常规ＥＬＩＳＡ缩短了３０－９０分钟。通过对猪旋毛虫病阴阳性血清检测，表明该方法敏感性为９３．６２％，特异性为９６．８８％。为猪旋毛虫病提供了一种快速诊断方法。     

贵妃鸡蛋重和繁殖率关系探讨

选２８￣２９周龄贵妃鸡新产种蛋，按蛋重分４组作蛋重和繁殖率关系试验。结果表明，蛋重４１￣４５克组受精率，孵化率和健雏率分别达９８．１１％，９４．２３％和９７．９６％，极显著高于蛋重３０克和３１￣３５克组，４５克内蛋重与三率呈强正相关，小于３０克重组三率极显著低于其它组（Ｐ〈０．０１），试验说明贵刀鸡种蛋最佳蛋重为４１￣４５克，小于３０克的蛋不宜作种用。     

贵州蜂胶品质的检测

对贵州三个不同地区的蜂胶样品检测，含蜡量≤３０％，杂质含量≤２０％，乙醇提取物含量〉５０％，酸值〈４０ｍｇＫＯＨ，碘值≤２０％，皂化值〈１００ｍｋＫＯＨ，过氧化值，０．８％，黄酮类化合物定笥反应均为（＋）反应。     

青海鹅观草和无芒鹅观草种子萌发条件及特性的研究

对产地，收获时间，贮藏条件相同的青海鹅观草和无芒鹅观草种子进行了１５、２０、２５、３０℃四种恒温及１０－２５、１５－２５、１５－３０、２０－３０℃四种变温处理，测定其萌发适宜温度；相继以适宜萌发温度，分别结合预冷，光照和０．２％硝酸钾三种预处理及低温、变温等措施测定两种子适宜的破除休眠技术。结果表明；鹅观草种子适宜萌发为１５－２５℃、１５－３０℃变温及２０℃恒温，青海鹅观草还适宜１０－２５℃的低变     

不同啃食强度对白沙蒿影响的初步研究

根据飞播区牲畜对白沙啃食程度的不同，采用人工模拟放牧方法对牲畜啃食的嫩枝及绿色部分设置了１０％、３０％、５０％、７０％、９０％、对照６种不同啃食强度的处理，探讨啃食强度对白沙蒿生长的影响。试验结果表明，白沙蒿放牧利用强度控制在３０％以下，有利于白沙蒿的生长，否则会影响其正常生长。     

抗旋毛虫循环抗原单克隆抗体的制备及初步应用

为评价单克隆抗体用于检测旋毛虫循环抗原（ＣＡ）的应用价值，作者利用淋巴细胞杂交瘤技术制备了４个抗旋毛虫ＣＡ的杂交瘤细胞系，建立了以单克隆抗体双抗体夹心ＥＬＩＳＡ检测ＣＡ的方法，并初步应用于动物血清中ＣＡ的检测。应用该法检测旋毛虫病猪血清的阳性率为７２．１％（３１／４３）。６０头健康猪、３０头感染猪囊虫和３０头感染弓形虫的猪均为阴性。对流行地区河南邓县３４头和湖北囊樊１３４头屠宰猪进行流行病学调查，     

秸秆饲料巧利用

秸秆饲料巧利用＠苏秀侠，邱山秸秆饲料巧利用苏秀侠，邱山我国农作物播种面积居世界第一位，秸秆产量达５．７亿吨／年，占全世界秸秆总产量的２０％－３０％。这是一种不容忽视的、巨大的饲料资源。目前，用秸秆作饲料的处理方法有三种。１．物理处理法：用机械对秸秆进行粉碎...     

种公猪精液品质与微量元素关系研究

对１４头种公猪精液品质下降原因进行了７种微量元素的检测分析，结果表明，检测组精液中Ｚｎ，Ｓｅ、Ｃｏ三种元素含量分别比对照组少４０．４％，５１．２％和３１．６％，两组比较差异达极显著水平和显著水平。Ａ组鬃毛中Ｚｎ、Ｓｅ、Ｃｏ三种微量元素含量分别比Ｂ组少４３．３％、４８．６％和２６％。其他４种微量元素含量差异不显著。结果还表明，种公猪精液品质下降与Ｚｎ、Ｓｅ、Ｃｏ三种微量元素含量偏低有直接关系。     

法氏囊病毒污染鸡场附近麻雀自然感染法氏囊病毒的初步调查

从传染性法氏囊病流行的鸡场捕杀麻雀３０只，用鸡传染性法氏囊病病毒单克隆抗体类心阻断ＥＬ１ＳＡ检测抗体，阳性检出率高为１０％（３／３０）；以逆转录一聚合酶链反应检测病毒核酸，阳性检出率为１３．３％；ＲＴ－ＰＣＲ阳性样本病毒分离亦为阳性。     

犬细小病毒酶标试剂盒的研制

用本研究室研制的犬细小病毒单克隆抗体和多克隆抗体，采用夹心ＥＬＩＳＡ原理，研制成功了犬细小病毒快速诊断试剂盒。对从临床病例中收集获得的４７份粪便样品分别用血凝试验和试剂盒进行了检测。两种方法的总符合率为９１．５％，对于明显阳性和阴性样品，两种方法符合率为１００％。试剂盒具有简便、快速、特异的优点。在３０分钟即可用肉眼判定结果。与ＣＤＶ、ＩＣＨＶ、ＦＰＶ、ＭＥＶ没有交叉反应。在４℃可保存６个月以上，     

3种禽支原体多重PCR检测方法的建立及应用

从鸡毒支原体、鸡滑液囊支原体和火鸡支原体 3种致病性支原体液体培养物中提取DNA,用特定引物分别和组合进行PCR,均得到了特异性扩增产物,片段大小分别为732、207和850 bp。直接用液体培养物进行PCR亦得到了一致的电泳条带。试验结果表明,建立的PCR方法可用于上述3种支原体临床感染的鉴别诊断。ELISA血清学检测和气管拭子PCR检测的比较结果表明,该PCR方法可用于临床MG检测。     

Detection of feline immunodeficiency proviral DNA in peripheral blood lymphocytes by the polymerase chain reaction.

Feline immunodeficiency virus (FIV) proviral DNA was detected by the polymerase chain reaction method (PCR). PCR products were detected by gel electrophoresis and ethidium bromide staining. The P-10, P-15 and P-24 regions of the gag gene of FIV were chosen as the target sequences for amplification, and three primer pairs were prepared. The PCR products subjected to amplification with each primer pair were found to possess sites of digestion by a restriction enzyme, as hypothesized. They did not react with feline leukemia virus (FeLV)-infected or feline syncytium-forming virus (FeSFV)-infected cell-derived DNA, and specifically amplified FIV-infected cell-derived DNA. FIV proviral DNA was detected by the PCR method with either primer pair (one-step amplification: single PCR) in DNA derived from peripheral blood lymphocytes (PBL) from 7 of 12 FIV antibody-positive cats. When PCR products in each of the 12 cats were subjected to a second amplification using the same primer pair (two-step amplification: double PCR), FIV proviral DNA was detected in all of the cats. When PBL samples collected from three cats that were negative and three that were positive in the single PCR were cultured for a few weeks in the presence of interleukin 2, FIV proviral DNA was detected in all six cats by the single PCR method. The results suggest that either the use of cultured PBL as the sample or the performance of the double PCR method enables simple and specific detection of FIV proviral DNA in PBL.     

猪细小病毒、猪伪狂犬病病毒和猪圆环病毒2型多重PCR检测方法的建立与应用

根据GenBank中已发表的猪细小病毒(porcine parvovirus,PPV)、猪伪狂犬病病毒(pseudorabies virus,PRV)和猪圆环病毒2型 (porcine circovirus type 2,PCV2)基因序列,对各病毒基因区进行同源性分析,确定PPV 的VP2、PRV的 gD、和PCV2的ORF2基因为各病毒的诊断靶序列,设计特异性引物,在建立各病毒单项PCR技术的基础上,优化多重PCR反应条件,建立了3种病毒的多重PCR技术,可同时扩增PPV 313 bp、 PRV 217 bp和PCV2 447 bp的特异性片段。用多重PCR技术与单项PCR技术对比检测试验证明两者的符合率为100％,表明建立的多重PCR检测方法,具有特异、快速、准确的特点,可同时鉴别诊断这3种病毒。从10个发病猪场和门诊病例的病猪采集的211份样品,用建立的多重PCR检测方法,检出PPV阳性42份,阳性率为19.91%;PRV阳性26份,阳性率为12.32%;PCV2阳性56份,阳性率为26.54%;2种以上病毒混合感染25份,混合感染阳性率为11.85%。检测结果表明,山西省猪群已感染这3种疫病。     

PRV、PCV-2、PPV多重PCR检测方法的建立及其应用

根据GenBank上已发表的猪伪狂犬病病毒(PRV)、猪Ⅱ型圆环病毒(PCV-2)、猪细小病毒(PPV)核苷酸序列,设计并合成能分别特异性扩增PRV、PCV-2、PPV的引物。经条件优化,建立了同时检测PRV、PCV-2、PPV的多重PCR方法,所扩增特异性产物片段大小分别为217bp、394bp、748bp。该方法不仅特异性强、敏感性高,还克服了对上述病原分别进行检测所带来的诸多弊端,为猪伪狂犬病、猪Ⅱ型圆环病、猪细小病的临床诊断和流行病学调查等研究提供了新手段。     

Establishment and Preliminary Application of Triple PCR to Identify Three Kinds of Conditional Pathogenic Bacteria

The aim of this study was to establish a simultaneous triple PCR detection method for Staphylococcus aureus (S.aureus),Pseudomonas aeruginosa (P.aeruginosa) and Klebsiella pneumoniae (K.pneumoniae).Three pairs of specific primers had been designed according to nuc gene of S.aureus,toxR gene of P.aeruginosa and PhoE gene of K.pneumoniae.The triple PCR reaction conditions were optimized on the basis of single PCR methods.At the same time,specificity,sensitivity and repeatability tests of the triple PCR method were studied,and the results of bacteria isolation and culture and the triple PCR method were compared.The results showed that the amplification product sizes were 484,278 and 368 bp,respectively.The optimal annealing temperature was 56 to 59 ℃,the concentrations of primers were all 0.2 μmol/L,dNTP concentration was 200 μmol/L,Mg²⁺ concentration was 2.5 mmol/L.The specificity test showed that there was no cross reaction between these three bacteria templates and other eight kinds of common bacteria templates,such as Bordetella bronchiseptica.The minimum of simultaneous detection of three bacteria genomic DNA was 10^-5 ng/μL.The results of three repeatability tests of triple PCR were the same which indicated the repeatability was good.30 samples from mice were detected by bacteria isolation and culture and the triple PCR method,the detection rate of triple PCR was slightly higher than the bacteria isolation and culture.The positive samples detected by bacteria isolation and culture were also positive detected by triple PCR.The results showed that a specific,sensitive and efficient triple PCR system had been established and could provide the technical support for bacteria detection and epidemiological investigation of laboratory animals.     

3种瘤胃纤维分解菌实时定量PCR方法的建立

为探索以减毒胞内侵袭菌介导的黏膜免疫对宿主动物胃肠道微生态的影响,本试验以白色瘤胃球菌、黄化瘤胃球菌和产琥珀酸丝状杆菌3种主要瘤胃纤维分解菌16S rRNA分别设计引物,以山羊瘤胃液提取细菌总DNA,分别扩增3种纤维分解菌目的DNA片段,并连接至pMD-18 T Vector上,经PCR和测序鉴定后,以不同稀释度的重组质粒为模板进行荧光定量PCR反应。结果显示,扩增得到的3种瘤胃纤维分解菌目的片段与已知菌种相应片段的同源性大于99%;以不同稀释度重组pMD-18 T为模板建立的荧光定量PCR扩增曲线差异明显,绘制标准曲线的相关系数均接近1,熔解曲线均呈单一峰值。因此,本试验成功建立了3种瘤胃主要纤维分解菌的实时定量PCR方法,为减毒胞内侵袭菌介导的黏膜免疫研究奠定了基础。瘤胃纤维分解菌;Real-time PCR;标准曲线;     

3种条件性致病菌三重PCR检测方法的建立及初步应用

本研究旨在建立一种能同时检测金黄色葡萄球菌(Staphylococcus aureus,S.aureus)、绿脓杆菌(Pseudomonas aeruginosa,P.aeruginosa)、肺炎克雷伯杆菌(Klebsiella pneumoniae,K.pneumoniae)的三重PCR检测方法.根据金黄色葡萄球菌nuc基因、绿脓杆菌toxR基因、肺炎克雷伯杆菌PhoE基因设计并合成引物,在单一PCR条件基础上优化建立三重PCR反应条件,并进行特异性、敏感性和重复性分析及与细菌分离培养的比对试验.结果显示,3对引物均能特异性扩增出目的条带,大小分别为484、278和368 bp.最佳退火温度在56~59 ℃之间,引物浓度均为0.2 μmol/L,dNTP浓度为200 μmol/L,Mg²⁺浓度为2.5 mmol/L.3种细菌间无交叉反应.对支气管鲍特杆菌等其他8种实验动物常见致病菌均无交叉反应.最低能同时检测到10^-5 ng/μL的细菌基因组DNA.3次重复结果一致,表明建立的三重PCR方法重复性好.同时采用细菌分离培养法和三重PCR方法对30份实验小鼠样本进行检测,对比结果显示三重PCR方法检出率略高于细菌分离培养法,细菌分离培养法呈阳性的样品,三重PCR方法均能检出.结果表明,本试验建立的三重PCR检测方法具有特异、敏感、高效等优点,为实验动物细菌快速检测和流行病学调查提供了技术支持.     

牛轮状病毒三种核酸检测方法的比较

利用针对牛轮状病毒（BRV）的普通PCR、实时荧光定量PCR（Real-time PCR）和环介导等温扩增技术（LAMP）三种核酸检测方法对不同质粒浓度的样品和417份临床疑似样品进行检测,比较三种核酸检测方法的检出结果。三种核酸检测方法中LAMP最敏感敏感,能检测到1拷贝/μL,Real-time PCR能检测到100拷贝/μL,普通PCR只能检测到1×104拷贝/μL。但结合临床样品检测表明：常规PCR方法敏感度低会造成一部分漏检,LAMP灵敏度高又会造成错检。综合比较三种方法后,推荐用LAMP结合real-time PCR,不仅节约成本,且结果更为准确可靠,可提高牛轮状病毒的检出率。     

Detection and identification of food-borne pathogens of the genera Campylobacter, Arcobacter and Helicobacter by multiplex PCR in poultry and poultry products

The objective of this study was to develop a multiplex polymerase chain reaction (PCR) to detect and differentiate food-borne pathogens of the three genera Campylobacter, Arcobacter and Helicobacter in a single step procedure. One common reverse primer and three genus-specific forward primers were designed by hybridizing to the 16S rRNA of selected reference strains. Besides the species with significance as food-borne pathogens isolated from poultry meat--Campylobacter jejuni, Campylobacter coli, Arcobacter butzleri and Helicobacter pullorum--several other members of these genera were tested to determine the specificity of the designed multiplex PCR. In total, 20 ATCC and NCTC reference strains of Campyobacter, Arcobacter and Helicobacter were used to evaluate the PCR. Specific amplificates were obtained from all thermophilic species of Campylobacter as well as from species of Arcobacter and Helicobacter. No amplification product was obtained from the non-thermophilic Campylobacter, C. hyointestinalis and C. fetus. Furthermore, a total of 43 field strains of the three genera isolated from poultry, pigs, cattle and humans were investigated using this PCR. To confirm the classification of 10 H. pullorum strains the 16S rRNAs were sequenced. The developed PCR is a helpful diagnostic tool to detect and differentiate Campylobacter, Arcobacter and Helicobacter isolated from poultry and poultry products.     

绵羊痘病毒、山羊痘病毒及羊口疮病毒多重PCR检测方法的建立和应用

为了建立鉴别绵羊痘病毒(SPPV)、山羊痘病毒(GTPV)和羊口疮病毒(ORFV)的多重PCR检测方法,针对GenBank中3种病毒的基因组序列,合成了3对引物,通过优化多重PCR反应条件,建立了鉴别检测3种病毒的多重PCR方法。特异性试验表明,应用该方法可分别扩增出3种病毒对应的目的片段,对大肠埃希菌、沙门菌、猪圆环病毒2型(PCV2)、猪细小病毒(PPV)、Vero细胞、正常羊组织的DNA和灭菌双蒸水均无扩增;敏感性试验表明,该方法最低检测量分别为30.46pg/μL的绵羊痘病毒、28.9pg/μL的山羊痘病毒和26.94pg/μL的羊口疮病毒基因组DNA;应用本方法对85份临床病料进行检测,结果与其他已建立的单项PCR检测方法结果一致,说明该方法可以用于临床上SPPV、GTPV和ORFV的鉴别诊断。