球虫分子生物学检测方法研究进展

球虫病是由球虫引起的一种发病率高、致死率低、显著影响动物生长,对畜牧业危害最严重的寄生虫病之一.建立高效的球虫检测方法对防控球虫病具有重要意义.为改善传统诊断方法的局限性,目前基于核酸的新兴诊断技术正在发展.论文综述了国内外球虫分子生物学检测方法的研究进展,分析了各种技术的优点和问题,展望了球虫分子生物学技术的应用前景...     

猪繁殖与呼吸综合征病毒的诊断及检测技术研究进展

猪繁殖与呼吸综合征是猪群中的一种免疫抑制性传染病,是严重危害养猪业的主要疫病之一,运用各种检测技术对该病做出准确的诊断是预防和控制该病的前提。猪繁殖与呼吸综合征的检测包括检测病毒、检测血清抗体及分型诊断,涉及的技术和方法有病毒分离技术、免疫组化技术、RT-PCR技术、间接免疫荧光试验、免疫过氧化物酶单层细胞试验、酶联免疫吸附试验、基因芯片技术等。每种诊断技术都有其各自的优势和局限性,应根据研究目的及工作条件等予以选择,作者对各种诊断技术的原理、方法及特点进行了概述和比较,并对今后的应用前景和发展趋势进行了展望。     

牛血清白蛋白泡沫法提取庆大霉素产物快速检测方法的研究

采用醋酸纤维薄膜电泳法分离庆大霉素与牛血清白蛋白,再用紫外分光光度法测定蛋白质泡沫法提取庆大霉素含量的方法.该方法中,选择620 nm作定量峰,庆大霉素和氨基黑10B的结合物浓度在1.25～10μg/mL范围内,吸收度与浓度的线性关系良好,相关系数为0.999 6,回收率为100.59%.     

血清对猪前脂肪细胞分化过程中聚脂相关基因表达的影响

为了探讨血清对猪前脂肪细胞诱导分化的影响,筛选更优的诱导方法.采用胶原酶消化法分离猪皮下前脂肪细胞,用含50 nmol·L~(-1)胰岛素、100 nmol·L~(-1)地塞米松、0.25 mmol·L~(-1)3-异丁基-1-甲基黄嘌呤、100nmol·L~1罗格列酮及添加(对照组)或不添加(试验组)10%FBS的分化培养液1和2对前脂肪细胞进行诱导分化,借助实时定量RT-PCR方法检测了细胞分化过程中聚脂相关基因PPARα、C/EBPα、FASN、ACOX1、GPAT和ENPP2的表达模式.结果显示:血清对PPARγ和FABP4两基因的表达有极显著的上调作用(P<＜0.01),而对其他6个基因的表达有极显著的下调作用(P＜0.01).试验组中FASN、GPAT和ACOX13个基因诱导分化各时间点的综合表达景均极显著高于对照组(P＜0.01),3基因表达量变化趋势间均达到极显著相关(P＜0.01).试验组中PPARα、PPARγ、C/EBPα和ACOX1 4个基因的基因表达最变化趋势间均达到显著或极显著正相关.研究表明:前脂肪细胞分化过程中,细胞内脂肪酸的生物合成和β氧化均在发生,细胞内脂肪含量积聚的快慢取决于2个途径力量的对比;PPARα、PPARγ、C/EBPα和ACOX14个基因间存在极强的协同表达现象;在细胞内脂肪积聚快慢上,含血清的分化培养液1要优于不含血清的分化培养液2.     

Description of outcomes of experimental infection with feline haemoplasmas: Copy numbers, haematology, Coombs’ testing and blood glucose concentrations

The aim of this study was to compare blood copy, haematological and glucose values between cats experimentally infected with either Mycoplasma haemofelis (Group HF: 10 cats), ‘Candidatus M. haemominutum’ (Group HM: 3 cats) or ‘Candidatus M. turicensis’ (Group TU: 3 cats). Blood samples were collected regularly up to 85 days post-infection (DPI) for haemoplasma real-time quantitative PCR, haematology, Coombs’ testing and blood glucose measurement. Statistical analysis was performed using a general linear model (ANOVA) appropriate for a repeated measures experiment with significance set as P < 0.05. Cats in Group TU had significantly lower blood copy numbers than cats in Group HF (P < 0.001) and HM (P < 0.001). All Group HF cats developed anaemia (often severe), macrocytosis and evidence of erythrocyte-bound antibodies whereas Groups HM and TU cats did not. Group HF had significantly lower PCVs, haemoglobin concentrations and red blood cell counts, and significantly higher mean cell volumes, than Groups HM and TU. In Group HF, erythrocyte-bound antibodies reactive at 4 °C (both IgM and IgG) appeared between 8 and 22 DPI and persisted for two to four weeks, whereas those reactive at 37 °C (primarily IgG) appeared between 22 and 29 DPI and persisted for one to five weeks. In most cats antibodies appeared after the fall in haemoglobin started. Although Group TU had significantly lower glucose concentrations than Groups HF (P = 0.006) and HM (P = 0.027), mean blood glucose concentrations remained within the reference range in all groups. This study demonstrates that M. haemofelis infection, in contrast to ‘Candidatus M. haemominutum’ and ‘Candidatus M. turicensis’ infection, can result in a severe macrocytic anaemia and the development of cold and warm reactive erythrocyte-bound antibodies.     

Detection of Bartonella spp. in wild rodents in Israel using HRM real-time PCR

The prevalence of Bartonella spp. in wild rodents was studied in 19 geographical locations in Israel. One hundred and twelve rodents belonging to five species (Mus musculus, Rattus rattus, Microtus socialis, Acomys cahirinus and Apodemus sylvaticus) were included in the survey. In addition, 156 ectoparasites were collected from the rodents. Spleen sample from each rodent and the ectoparasites were examined for the presence of Bartonella DNA using high resolution melt (HRM) real-time PCR. The method was designed for the simultaneous detection and differentiation of eight Bartonella spp. according to the nucleotide variation in each of two gene fragments (rpoB and gltA) and the 16S–23S intergenic spacer (ITS) locus, using the same PCR protocol which allowed the simultaneous amplification of the three different loci. Bartonella DNA was detected in spleen samples of 19 out of 79 (24%) black rats (R. rattus) and in 1 of 4 (25%) Cairo spiny mice (A. cahirinus). In addition, 15 of 34 (44%) flea pools harbored Bartonella DNA. Only rat flea (Xenopsyla cheopis) pools collected from black rats (R. rattus) were positive for Bartonella DNA. The Bartonella sp. detected in spleen samples from black rats (R. rattus) was closely related to both B. tribocorum and B. elizabethae. The species detected in the Cairo spiny mouse (A. cahirinus) spleen sample was closely related to the zoonotic pathogen, B. elizabethae. These results indicate that Bartonella species are highly prevalent in suburban rodent populations and their ectoparasites in Israel. Further investigation of the prevalence and zoonotic potential of the Bartonella species detected in the black rats and the Cairo spiny mouse is warranted.     

变性高效液相色谱检测技术在微生物领域的应用

变性高效液相色谱法（DHPLC）是一种高通量筛选DNA序列变异的新技术。本文从该技术的工作原理、主要特点以及其在耐药检测、基因分型和鉴定、混合微生物分离鉴定等微生物领域的应用方面作了较详细的综述。     

贵州白香猪的遗传检测

选取27个微卫星位点,应用PCR技术对贵州白香猪的2个品系进行遗传质量监测。结果在所选的27个微卫星位点中,共有24个位点获得稳定的扩增结果,其中SW911位点与S0026两个位点在群体内表现为单态纯合。2个品系有效等位基因、平均多态信息含量、平均杂合度、基因纯合率和平均近交系数分别为1.8310、1.7951、0.3436、0.3249、0.4329、0.4188、52.63%、58.55%、0.5377、0.5605。结果表明微卫星标记可用于检测贵州白香猪的遗传质量,也可用于分析封闭群的遗传多态性。是一种有潜力的试验动物遗传学检测标记。     

氟喹诺酮类药物残留免疫学检测方法的研究

以沙拉沙星(SALX)为半抗原,牛血清白蛋白(BSA)偶联后免疫兔,获得对多种氟喹酮类(FQNS)药物有特异性的广谱性抗体.以SALX与卵清白蛋白(OVA)的偶联物作包被原,建立并优化了间接竞争ELISA检测方法,对SALX最低检出限达19.319 μg/L,且对诺氟沙星、环丙沙星和恩诺沙星均有特异性识别,最低检测限分别是22.646、26.181和19.054 μg/L.结果表明,该抗体可用于多种氟喹诺酮类药物残留筛选检测.     

猪链球菌2型四川分离株毒力基因的检测及其序列分析

根据猪链球菌2型的MRP、EPF和SLY基因设计合成了四对引物,对我们分离鉴定的2005四川分离株进行毒力因子检测,并对阳性产物测序比较。结果以分离株为模板,获得了与预期结果一致的867bp(MRP)、572bp(EF)的2个片段,以及SLY的长度为1502bp目的片段(外引物)和1443bp的目的片段(内引物)。说明分离株SSsc0501为MRP+EF+SLY+,属于具有3种主要毒力因子的强毒株。经对PCR阳性产物测序及序列分析,结果其与P1/7株的核苷酸序列完全一致,与1933株的同源性达99.7%,仅在128位、217位、744位、1380位、1386位的5个核苷酸发生突变。在氨基酸水平上SSsc0501株与1933株的同源性达99.6%,有2个氨基酸发生替代。