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《中国动物传染病学报》2015,(6)
本研究通过设计、合成并修饰基因特异性引物和寡核苷酸探针并标记生物素,利用该探针与荧光编码微球偶联后,与抽提的对虾白斑综合征病毒(White spot syndrome virus,WWSV)的PCR产物进行杂交反应,用液相芯片检测仪(Luminex200)检测荧光信号建立了WWSV快速液相芯片检测方法。该方法具有较好的特异性,偶联特异性探针的微球只与相应的病毒基因的PCR产物反应,而不与其他虾病病毒基因反应,可以检测到107的稀释度。本研究初步建立的WSSV液相芯片检测方法为进一步建立几种虾病同时快速检测奠定了基础。 相似文献
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为建立检测鲤春病毒血症病毒（SVCV）的液相芯片快速检测技术,用DNAStar软件对GenBank中SVCV的G基因进行序列分析,设计SVCV特异性引物和探针并标记生物素,偶联荧光编码微球后与SVCV病毒RT—PCR产物杂交反应,用液相芯片仪器检测荧光信号。结果表明液相芯片检测体系能够正确的检测出SVCV。病毒核酸的最低检出量为10pg,检测特异性高,初步建立了检测SVCV的液相芯片技术,为进一步构建其他水生动物病原体全新快速高通量检测平台奠定基础。 相似文献
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为建立禽流感病毒和新城疫病毒的液相芯片快速检测方法,试验设计并合成针对H5、H9亚型禽流感病毒HA基因以及新城疫病毒NP基因的特异性引物、探针及探针反向序列,并将下游引物和探针反向序列进行生物素标记。将探针与不同编号的荧光编码微球偶联。通过将探针-荧光编码微球偶联物与HA、NP基因的PCR产物杂交反应,用液相芯片检测仪检测荧光信号建立禽流感和新城疫快速液相芯片检测方法。结果显示,该法具有较高的特异性和灵敏度,非特异性反应很小,无明显交叉反应,该液相芯片快速高通量检测方法可同时检测新城疫病毒、H9亚型和H5亚型禽流感病毒,三株病毒毒株抗原的检测灵敏度分别为10~(-4)、10~(-5)和10~(-5)。同时,检测结果显示:三个毒株同步检测与单个毒株分别检测在检测的灵敏上度基本相当。研究建立的可以同时检测新城疫病毒、H9亚型和H5亚型禽流感病毒的快速高通量液相芯片方法,为该类病毒的检测提供了一种新工具,同时也为其他类似病毒的检测提供了借鉴。 相似文献
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用DNAStar7.0软件对GenBank中IHNV的N基因进行序列分析,设计IHNV特异性引物并标记生物素,将探针氨基化修饰,与荧光编码微球偶联后与RT-PCR产物杂交反应,用液相芯片仪器检测荧光信号,建立了传染性造血器官坏死病毒（IHNV）的液相芯片快速检测技术。结果表明液相芯片检测体系对IHNV病毒核酸的最低检出量为100pg,且特异性高,与其他病毒无交叉反应。应用建立的液相芯片技术检测鱼类中IHNV,并与RT-PCR方法进行比较,检测结果基本一致,但该方法比RT-PCR法更灵敏。表明初步建立了检测IHNV的液相芯片技术,为进一步构建其他水生动物病原体快速高通量检测平台提供参考。 相似文献
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《中国动物传染病学报》2017,(2)
本研究通过设计、合成并修饰基因特异性引物和寡核苷酸探针并标记生物素,利用该探针与荧光编码微球偶联后与虾黄头病毒(Yellow head virus,YHV)、虾桃拉综合征病毒(Taura syndrome virus,TSV)和白斑综合征病毒(White spot syndrome virus,WSSV)的PCR产物杂交反应,用液相芯片检测仪(Luminex200)检测荧光信号,初步建立了虾黄头病毒、桃拉病毒和白斑病毒的快速同步检测方法。结果显示,建立的该方法具有较好的特异性,偶联特异性探针的微球只与相应的病毒基因的PCR产物反应,而不与其他虾病病毒基因反应,YHV、TSV和WSSV检测灵敏度高分别为401.5、251.5和75.2 copies。 相似文献
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为建立小反刍兽疫病毒(PPRV)液相芯片检测方法,根据Gen Bank上公布的PPRV的N蛋白基因序列高度保守区设计引物与探针;对探针进行C12臂修饰后与荧光编码微球偶联,将偶联后的探针与PPRV的PCR产物进行杂交,在Bio-Plex系统上读取荧光信号,建立了小反刍兽疫病毒液相芯片检测方法,并对该方法的特异性和敏感性进行了研究,最终用建立的方法对临床样本进行检测。结果显示,该方法只与PPRV基因的PCR产物反应,而不与其他病毒基因反应,可检出的最低基因拷贝数为5. 78×104copies/m L,表明该方法具有良好的特异性与敏感性。利用该方法对25份临床样本进行检测,共检出阳性样本15份,与传统PCR方法检测结果一致。本研究建立了小反刍兽疫病毒的快速液相芯片检测方法,为进一步建立多种外来动物疫病的液相芯片检测方法奠定了基础。 相似文献
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4种重要虫媒病的核酸液相芯片高通量检测方法的建立 总被引：1，自引：1，他引：0
为建立可检测鹿流行性出血热病毒(EHDV)、阿卡斑病毒(AKV)、蓝舌病病毒(BTV)和水泡性口炎病毒(VSV)的液相芯片快速检测技术,用DNAStar软件对GenBank中BTV的VP7基因、EHDV的VP7基因、AKV的N基因和VSV的NP基因序列进行序列分析,设计针对这些基因的特异性探针并标记生物素,分别与不同编号的荧光编码微球偶联后再与这些病毒相应基因的PCR产物杂交反应,用液相芯片检测仪(Liquichip 200)检测荧光信号建立了以上4种虫媒病的快速液相芯片检测方法。检测结果显示,该方法具有较好的特异性,偶联特异性探针的微球只与相应的病毒基因的PCR产物反应,而不与其他虫媒病病毒反应;检测灵敏度达到50~100个TCID50。本研究建立了可以同时检测鹿流行性出血热病毒、阿卡斑病毒、蓝舌病病毒和水泡性口炎病毒的快速高通量液相芯片技术,为其他类似病毒的快速高通量检测提供了借鉴和经验。 相似文献
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为建立可检测鹿流行性出血病病毒(EHDV)的液相芯片快速检测技术,用DNAStar软件对GenBank中EHDV的VP7基因进行序列分析,设计EHDV特异性探针并用生物素标记,与荧光编码微球偶联后与病毒VP7基因的PCR产物杂交反应,用液相芯片检测仪(Liquichip200)检测荧光信号建立了EHDV快速高通量液相芯片检测方法。检测结果显示,该法具有较好的特异性,不与其他虫媒病病毒反应;检测灵敏度为100个TCID50。建立了快速检测EHDV的液相芯片技术,为进一步搭建EHDV快速高通量检测平台奠定了基础。 相似文献
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[目的] 建立空肠弯曲菌液相芯片检测方法。[方法] 本研究将空肠弯曲菌HIP蛋白单克隆抗体与聚苯乙烯微球偶联,结合双抗体夹心技术建立空肠弯曲菌液相芯片检测方法,测定不同浓度的抗体与微球偶联率。通过L9（34）正交设计试验优化方法的反应条件,并进行灵敏度和特异性试验。[结果] 较优实验条件即多克隆抗体工作浓度为1:100、生物素标记的羊抗兔IgG工作浓度为1:500、SA-PE的工作浓度为10ug/mL、生物素标记的抗体与SA-PE反应时间为90min。该方法灵敏度可达103CFU/mL,与其他常见食源性致病菌无交叉反应。[结论] 该方法灵敏度高、特异性强、重复性好,可快速检测食品中的空肠弯曲菌。 相似文献
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Identification of subpopulations of bovine mammary-gland phagocytes and evaluation of sensitivity and specificity of morphologic and functional indicators of bovine mastitis 总被引：2，自引：1，他引：1 
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下载免费PDF全文 A. L. Rivas R. Tadevosyan F. W. Quimby T. Coksaygan D. H. Lein 《Canadian journal of veterinary research》2002,66(3):165-172
The number and function of bovine mammary-gland phagocytes were assessed in 8 lactating cows, each tested at least twice within an 8-mo period (total number of observations, 20). Macrophages and polymorphonuclear (PMN) cells were evaluated by conventional cytology, flow cytometry, fluorescent microscopy, and somatic-cell count (SCC). Phagocytosis was evaluated from the uptake of fluorescent beads and expressed as median fluorescence intensity (MFI). Two major subpopulations of phagocytes, of low or high MFI (LFI or HFI), were observed, and there were up to 4 sub-subpopulations within the HFI subpopulation of both macrophages and PMN cells. Fluorescent microscopy identified phagocytes containing up to 4 beads per cell. Cows showing ≤ 72.3% phagocytes by cytology were regarded as non-mastitic (11 observations), and those showing ≥ 80.7% phagocytes were considered to be mastitic (8 observations). Phagocyte MFI was negatively associated with mastitis; that is, the higher the MFI, the lower the SCC. The percentage of HFI PMN cells was the only indicator of mastitis with 100% sensitivity and specificity. Thus, bovine mammary-gland phagocytes consist of several subpopulations of different phagocytic ability, whose assessment more adequately predicts bovine mastitis than do morphologic indicators. 相似文献
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用AllGlo探针检测对虾白斑综合征病毒,阳性反应呈典型的S曲线,说明本检测探针和引物设计合理。经灵敏度测定,当样品DNA浓度低至100拷贝/μL时,仍可以有效扩增,但在103/μL之上时结果最佳。同时,经过与传统探针检测对比试验得出:用Taqman标记的探针检测相同量的样品时,AllGlo MAR标记的探针(FAM通道)CT值与FAM标记的探针的CT值没有明显区别,但是MAR标记的探针的荧光信号强度要比FAM标记的探针约高40%,更适合于对虾白斑综合征病毒的检测。 相似文献
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鹅细小病毒荧光定量PCR检测方法的建立及应用 总被引：1，自引：0，他引：1
利用已经分离得到的小鹅瘟病毒GPVSP株,设计合成1对特异性的引物和TaqMan探针,通过特异性试验、敏感性试验建立了GPVTaqMan荧光定量PCR检测方法。同时饲养产蛋种鹅,接种小鹅瘟病毒,在不同的时间段采取病料,并且利用荧光定量PCR检测病毒的存在与含量,得到了小鹅瘟病毒在产蛋种鹅体内的定植规律。 相似文献
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OBJECTIVE: To determine the in vitro elution characteristics of gentamicin from Plaster of Paris-gentamicin (POP-gent) beads. STUDY DESIGN: In vitro, controlled, experimental study. METHODS: The POP-gent beads were made using a bead mold from 20 g calcium sulfate hemihydrate, 5 mL (500 mg) gentamicin solution, and 3 mL of phosphate buffered saline (PBS). Control beads were made similarly, using 30 g of dried powder and 8 mL of PBS. Beads were left in the mold overnight, gas-sterilized with ethylene oxide, and stored at room temperature for 5 months before testing. Bead chains were placed in sterile tubes containing porcine serum, and tubes were placed in a 37 degrees C incubator on a rocker. Serum was removed at intervals over 14 days and the concentration of gentamicin determined by fluorescent polarization immunoassay. Serum antibacterial activity was determined against an equine origin Escherichia coli. RESULTS: POP-gent beads released gentamicin for the 14-day sampling period. Eighty percent of the gentamicin incorporated in the beads was released over the first 48 hours. Eluent from POP-gent beads inhibited the growth of E coli at all time periods. No gentamicin was eluted from control beads and control eluent did not inhibit growth of E coli. CONCLUSIONS: In this experimental model, POP-gent beads released bactericidal drug for 14 days. Eighty percent of the gentamicin incorporated into the beads was released during the first 48 hours. The drug retains its bactericidal activity after ethylene oxide sterilization and storage at room temperature for up to 5 months. CLINICAL RELEVANCE: Pop-gent beads may be a useful repository device to deliver gentamicin locally in tissues. 相似文献
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Streptococcus uberis is a significant cause of bovine mastitis throughout the world. Previous work from our laboratory demonstrated that S. uberis adhesion molecule (SUAM) is an important factor in adherence to and internalization of S. uberis into bovine mammary epithelial cells. Antibodies directed against SUAM significantly reduced bacterial adherence to and internalization into bovine mammary epithelial cells implying that SUAM is surface exposed. Objectives of this research were to: (1) predict surface exposed peptides, and (2) select peptide sequences for production of synthetic peptides with the final aim of evaluating their role in adherence and internalization and immunogenic potential. The Kyte/Doolittle hydropathicity prediction method; Chou/Fasman β-turn prediction method; and output from Coils, Paircoil and MultiCoil scores for prediction of secondary and tertiary structures were used. Prediction algorithms resulted in identification of five overlapping regions of the SUAM sequence with the most hydrophilic valleys and the highest peaks for β-turns. The five 15-mer SUAM epitopes selected by bioinformatic analysis were produced to evaluate the immunogenic value and pathogenic role of these putative domains. Peptides were bound to fluorescent latex beads, incubated with MAC-T bovine mammary epithelial cells, and internalization into MAC-T cells was evaluated using confocal laser and transmission electron microscopy. All peptides evaluated induced some degree of internalization of fluorescent beads into MAC-T cells; however, 2 peptides induced significantly more internalization of fluorescent beads than the other peptides evaluated. These peptides, designated III and IV, were located in the central region of SUAM, between two coiled-coil regions. Convalescent sera were tested against these biotinylated peptides for SUAM specific immune response using an indirect ELISA format. Among the 5 peptides evaluated, peptides I, II and V elicited significant serological response suggesting that the N-terminal region (peptide I), central region (peptide II) and C-terminal region (peptide V) are immunodominant epitopes of SUAM. Results will be useful to design immunotherapeutic tools based on immunodominant epitopes. 相似文献
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M J Daley E R Oldham T J Williams P A Coyle 《American journal of veterinary research》1991,52(3):474-479
Polymorphonuclear cells have a critical role in the pathogenesis of bovine mastitis. We have documented that experimentally induced Staphylococcus aureus mastitis is associated with cyclic increase and decrease in the quantity of viable bacteria shed in the milk. Concomitant with this cycling of bacteria is an inverse cycling of the hosts cells within the milk. Such somatic cells were determined to be greater than or equal to 95% polymorphonuclear cells. The quality of these cells was evaluated by measuring their relative efficiency of bacterial killing and phagocytosis at various times during an infection. Host polymorphonuclear cells had as much as 10,000-fold variation in the bactericidal failure rate for staphylococci during cell cycling. The most efficient bactericidal effect was observed at or near the peak of the somatic cell count (SCC). The ability of these cycling cells to ingest fluorescent beads was also quantitated by use of flow cytometry. The percentage of phagocytic polymorphonuclear cells that ingested fluorescent latex beads ranged from 15 to 80% of the total cell population during cell cycling, and tended to be optimal at or near peak SCC. In addition, the average number of beads ingested varied between 1 and 2 particles/polymorphonuclear cell, with as many as 17% of the phagocytic cells ingesting 4 or more beads at maximal efficiency. Polymorphonuclear cells from quarters infected with S aureus varied quantitatively (total SCC) and qualitatively (bactericidal activity and phagocytic ability) during the course of an infection. Not only is the quantity of host's phagocytic cells in the mammary gland central to the defense mechanism against infection, but the biological activation state appears to be equally important. The role of these cells in the pathogenesis of a cycling infection is presented in a model to explain the cyclic nature of mastitis. 相似文献
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Aylin Atilla VMD MS Harry W. Boothe DVM MS Diplomate ACVS Melissa Tollett BS Sue Duran BS MS PhD Dubraska Campos Diaz DVM MS Jameson Sofge BS Dawn M. Boothe DVM PhD Diplomate ACVIM & ACVCP 《Veterinary surgery : VS》2010,39(6):715-721
Objective: To describe in vitro elution characteristics of amikacin and vancomycin from calcium sulfate hemihydrate 98% (plaster of Paris, POP) beads and characterize eluent inhibition of Staphylococcus spp. Study Design: Experimental study. Methods: POP beads were impregnated with amikacin or vancomycin alone or in combination and then incubated alone or in combination for 84 days at 37°C in plastic tubes containing sterile phosphate‐buffered saline (PBS). Beads containing no antimicrobial served as negative control. Beads were intermittently moved to a new tube containing drug‐free PBS. Antimicrobial was measured in the eluent using a polarized fluorescent immunoassay. Eluent inhibition of Staphylococcus spp. was determined at each time point. Results: Antimicrobial release from beads was characterized by an initial rapid phase then a slower phase. Although antimicrobial release from beads occurred throughout the 84 days, most was in the first 24 hours, except for vancomycin alone. Duration of eluent inhibition of Staphylococcus spp. growth ranged from 0.5 (amikacin alone) to 56 days (vancomycin alone). Control eluent did not inhibit bacterial growth. Conclusions: Amikacin elution from POP beads was rapid, inhibiting growth for <24 hours with or without vancomycin. Vancomycin elution was slower and inhibited growth for 56 days alone or for 5 days with amikacin. Clinical Relevance: Vancomycin‐impregnated beads appear to be reasonable as a therapeutic option whereas amikacin‐impregnated POP beads and amikacin and vancomycin combinations may require further study before considering as a therapeutic option. 相似文献
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Tamara B. Wills Allison M. Heaney K. Jane Wardrop Gary J. Haldorson 《Veterinary clinical pathology / American Society for Veterinary Clinical Pathology》2009,38(4):437-442
Background: Increased concentrations of circulating endothelial cells (CECs) are thought to be a biomarker of vascular injury in human patients with cardiovascular disease, neoplasia, vasculitis, sickle cell anemia, shock, and sepsis. Immunomagnetic isolation is a technique currently used to enumerate human CECs and can detect low numbers of cells. Objectives: The purpose of this study was to determine whether a standard protocol for immunomagnetic isolation could be used to obtain and enumerate CECs and a subpopulation of endothelial progenitor cells (EPCs) from canine whole blood. Methods: Cultured canine aortic endothelial cells were stained immunohistochemically with von Willebrand factor to verify morphology and number. Using magnetic beads conjugated with anti‐CD146, CECs/EPCs were isolated in culture and in canine whole blood. CD146‐positive cells were stained with fluorescein‐conjugated Ulex europaeus agglutinin 1 (UEA‐1) to confirm endothelial origin and cells were counted manually using a fluorescent microscope. The method was then applied to EDTA‐anticoagulated whole blood samples from 10 healthy client‐owned dogs. Results: The anti‐CD146–coated magnetic beads (>5/cell) bound the cultured canine aortic endothelial cells. Only rare UEA‐1–positive cells were obtained from whole blood, while >85–90% of cultured canine aortic endothelial cells were UEA‐1 positive. The percentage recovery of cultured canine aortic endothelial cells was >86%. CECs in canine whole blood had >8 beads attached to the surface and were 10–40 μm in size. Using immunomagnetic isolation, 43.4 ± 15.6 CECs/mL (range 24–70/mL) were isolated from canine whole blood samples. Conclusions: Immunomagnetic isolation is an acceptable method for enumerating canine CECs/EPCs in whole blood. Further studies are warranted to evaluate the clinical significance of CEC/EPC concentration in different canine diseases. 相似文献
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Bacillus subtilis-based direct-fed microbials augment macrophage function in broiler chickens 总被引：1，自引：0，他引：1
Lee KW Li G Lillehoj HS Lee SH Jang SI Babu US Lillehoj EP Neumann AP Siragusa GR 《Research in veterinary science》2011,91(3):e87-e91
The present study was conducted to evaluate the function of Bacillus subtilis-based direct-fed microbials (DFMs) on macrophage functions, i.e., nitric oxide (NO) production and phagocytosis in broiler chickens. DFMs used in this study were eight single strains designated as Bs2084, LSSAO1, 3AP4, Bs18, 15AP4, 22CP1, Bs27, and Bs278, and one multiple strain DFM product (Avicorr™) containing equal amount of Bs2084, LSSAO1 and 15AP4. NO concentrations were monitored in plasma and in the supernatants from the peripheral blood-derived monocytic cells (PBMC)-derived macrophages stimulated by either chicken recombinant interferon gamma (IFNγ) or lipopolysaccharide (LPS) from Escherichia coli or Salmonella typhi. In addition, phagocytosis of fluorescent beads and green fluorescent protein (GFP)-labeled Salmonella by PBMC-derived macrophage was assayed. Plasma NO levels were significantly higher in groups given 3AP4 or Bs27 diets compared with the control group at days 7 and 14. NO production by PBMC-derived macrophages stimulated with IFNγ or LPS was apparent, although the effect was strain-dependent. Phagocytosis of fluorescent beads or GFP-labeled Salmonella by macrophages was augmented in groups on DFM-supplemented diets compared with those fed the control diet. This study describes the immunomodulatory effects of Bacillus-based DFMs on innate immunity in broiler chickens. 相似文献