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免疫印迹(Westernblot)分析试验感染肝片吸虫和大片吸虫绵羊的抗片形吸虫分泌捧泄产物(FhESP和FgESP)IgG应答,结果表明,FhESP中分子质量在17.1~21.6ku之间的4条主要蛋白带仅被肝片吸虫感染以后(0WPI以后)的血清识别且不被对照组血清识别;FgESP中分子质量在19.0~71.1ku之间的6条主要蛋白带仅被大片吸虫感染以后(0WPI以后)的血清识别且不被对照组血清识别。 相似文献
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为建立更敏感、特异的牛片形吸虫病早期诊断方法,本试验将收集的大片吸虫分泌排泄产物(Fasciola gigantica excretory-secretory products,FgESP)进行凝胶过滤层析并从中筛选出检测效果最优的UV280吸收峰,从此峰对应的组分中筛选出诊断效果最优的层析组分后将其作为抗原,通过棋盘滴定试验优化抗原包被浓度、血清稀释度、酶标二抗稀释度、显色时间等,确定临界值,建立牛片形吸虫病间接ELISA诊断方法。对建立的方法进行敏感性和特异性试验,检测试验感染0~14周的水牛血清和临床160份水牛血清样本,并与已有的诊断抗原FgESP(阴阳临界值为0.320)进行诊断效果比较。结果显示,层析组分F22具有较优的检测效果。间接ELISA的最佳反应条件为:F22抗原包被浓度0.157 μg/mL,待测血清与酶标二抗的稀释度分别为1:400和1:40 000,显色时间为25 min。应用建立的方法检测15份水牛阴性血清,确定D450 nm阴阳性临界值为0.408。比较F22与FgESP发现,二者特异性相似,但F22作为抗原的敏感性高于FgESP。检测试验感染大片吸虫的水牛0~14周血清,结果表明,从感染第2周开始F22-IgG水平极显著高于FgESP-IgG水平(P<0.01),因此,F22比FgESP诊断效果更优。对160份水牛血清检测发现,F22阳性检出率为71.25%,FgESP阳性检出率为63.75%。上述结果表明,相比于FgESP,以F22作为诊断抗原建立的大片吸虫病间接ELISA诊断方法敏感性更高,可更有效地进行牛大片吸虫病的早期诊断。 相似文献
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目的大片吸虫cDNA文库的构建及组织蛋白酶L1(Fasciola gigantica Cathepsin L1,FgCL1)基因的克隆。方法提取大片吸虫成虫总RNA,运用SMART技术构建大片吸虫cDNA文库。根据文献设计并合成PCR引物,从上述文库中克隆大片吸虫FgCL1基因,定向克隆至原核表达载体pET28a中。结果文库容量为2.08×106pfu/mL,重组率为98%。扩增后的文库滴度为6.23×109 pfu/mL,插入片段平均大小约为1 000 bp。应用两个已知基因(FgCL1 FgGST)从文库中成功钓取到相应的全长基因。将含有FgCL1的阳性克隆测序,用Genebank Blast进行序列比较,证实为FgCL1基因。结论成功构建了cDNA文库,提供筛选大片吸虫基因资源;从该文库中克隆出FgCL1,为进一步表达该基因,研制诊断试剂盒创造了条件。 相似文献
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Molloy JB Anderson GR Fletcher TI Landmann J Knight BC 《Veterinary parasitology》2005,130(3-4):207-212
A commercially available ELISA for detecting antibodies to liver fluke was evaluated for use in Australia. Milk and serum samples from cattle and sheep in which infection with Fasciola hepatica was confirmed by detection of eggs in faeces were used to estimate sensitivity. Similar samples collected from cattle and sheep outside the F. hepatica-endemic area were used to estimate specificity. The ELISA was also evaluated for detecting antibodies to F. hepatica in milk from sheep and antibodies to Fasciola gigantica in sera from cattle and buffaloes, but with small numbers of samples. In cattle, the sensitivity and specificity of the ELISA were 98.2% and 98.3% using serum and 97.7% and 99.3% using milk. In infected herds, 41.4% and 41.5% of animals were positive in the serum and milk ELISAs, respectively, whereas F. hepatica eggs were found in faecal samples from 26.5% of animals. In sheep, the sensitivity of the ELISA was 96.9% and the specificity was 99.4%. In infected flocks, 60.2% of animals were positive in the serum ELISA and F. hepatica eggs were found in faecal samples 52.2% of animals. There was perfect agreement in the ELISA between paired serum and milk samples collected from ewes. The assay detected antibodies in sera from cattle and buffaloes with natural and experimental F. gigantica infections. In the experimentally infected animals, antibodies were detected 2 weeks post-infection. We conclude that the ELISA will be a valuable tool for diagnosing F. hepatica infections in cattle and sheep. The assay may also be useful for diagnosing F. gigantica infections but further studies are required to establish sensitivity and specificity. 相似文献
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水牛实验感染大片吸虫及ELISA检测特异性抗体的变化 总被引:3,自引:0,他引:3
目的 本试验为了对水牛感染大片吸虫的情况进行了解。方法 选 10头 6月龄水牛 ,分成 2组 ,每组 5头 ,A组对照 ,B组试验 ,每头经口感染 2 5 0个大片吸虫囊蚴。结果 水牛感染大片吸虫后的收虫率为 16 .2± 8.0 % ,在感染后第 13~ 14周检出虫卵。抗大片吸虫分泌排泄产物 Ig G水平从感染后第 2周明显升高 ,17周时达到高峰。结论 试验证实水牛对大片吸虫感染很敏感 ,EL ISA检测特异性抗体用于片形吸虫病的早期诊断有一定的意义 相似文献
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应用LD-PCR法构建大片吸虫成虫cDNA表达文库 总被引:3,自引:1,他引:2
为构建大片吸虫成虫cDNA表达文库,用Trizol试剂提取大片吸虫成虫总RNA,经反转录合成cDNA第一链,应用LD-PCR扩增方法,合成双链cDNA。用SfiⅠ内切酶修饰此双链cDNA,使形成两端分别带有SfiⅠA和SfiⅠB的黏性末端。经CHROMA SPIN-400柱纯化,收集400 bp以上的双链cDNA片段,将其连接于带有SfiⅠA和SfiⅠB末端的λTriplEx2噬菌体载体,经体外包装后,以XL1-Blue为受体菌构建cDNA表达文库。经测定,库容量为1.08×106PFU/mL,重组率为96.6%。扩增后的文库滴度为2.41×109PFU/mL,插入片段平均大小约为1 000 bp。这些结果表明已成功构建大片吸虫成虫cDNA表达文库,适合进一步筛选大片吸虫新基因。 相似文献
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取广西牛源大片吸虫成虫制备出分泌排泄(ES)抗原,用SDS-PAGE电泳,考马斯亮蓝染色显出7条蛋白带。免疫印迹试验(EITB)测出其主要特异性免疫成分分子量为36.3,28.5及25.5kDa,其中28.5与25.5kDa与法国肝片吸虫ES的部分抗原成分相同。两种片形吸虫与抗血清可以交叉使用无特异性,但与日本血吸虫抗原及抗血清均无交叉反应,人工感染大片吸虫2周后的兔血清即能识别此抗原。ES抗原提取方法简便,特异性好,敏感性高,可用于片形吸虫病免疫诊断。 相似文献