同时监测8种牛常见病原体的GeXP高通量快速检测技术

发布时间：2018-03-07 11:19

本文选题：牛传染病　切入点：GeXP　出处：《畜牧兽医学报》2017年10期 　论文类型：期刊论文

【摘要】：本研究旨在建立一种可同时检测8种常见牛传染病病原体(口蹄疫病毒、蓝舌病病毒、水疱性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒大肠杆菌、牛传染性鼻气管炎病毒和小反刍兽疫病毒)的GeXP高通量快速检测方法。将多重PCR技术和毛细管电泳技术有效地结合,根据上述8种病原体的基因保守序列,设计并合成了8对特异性引物,在每对特异性引物的5′端加上一段通用引物,形成特异性嵌合引物。优化反应体系和反应条件,用单一病毒和混合病毒样品来验证所建立的GeXP方法的特异性,用克隆质粒稀释液来测试GeXP方法的灵敏度,检测305份临床样品,进一步验证该方法的可靠性。结果:优化后的GeXP检测体系,可扩增出各病原体对应的特异片段,能在100拷贝·μL~(-1)水平同时并特异地检测出8种牛病病原体核酸。305份临床样品的检测结果与荧光PCR检测结果一致,阳性样品测序结果表明无非特异性扩增的假阳性。结果显示,所建立的GeXP方法具有高通量、特异性好、灵敏度高、方便快速等优点,可用于临床鉴别诊断和流行病学调查,具有较高的临床应用价值。
[Abstract]:The purpose of this study was to establish a novel pathogen for simultaneous detection of 8 common bovine infectious diseases (foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis virus, bovine viral diarrhea virus, bovine rotavirus, enterotoxigenic Escherichia coli). High throughput GeXP rapid detection method for bovine infectious rhinotracheitis virus and small ruminant ruminant virus. The multiplex PCR technique and capillary electrophoresis technique were effectively combined, according to the gene conserved sequence of the above eight pathogens, Eight pairs of specific primers were designed and synthesized. A segment of universal primers was added to the 5 'end of each pair of specific primers to form a specific chimeric primer. The reaction system and reaction conditions were optimized. Single virus and mixed virus samples were used to verify the specificity of the established GeXP method. The sensitivity of the GeXP method was tested with clone plasmid dilution solution, and 305 clinical samples were detected. Results: the optimized GeXP detection system can amplify the specific fragments corresponding to each pathogen. At the level of 100 copies 路渭 L ~ (-1), nucleic acid of 8 kinds of bovine disease pathogens, nucleic acid, was detected at the same time and the results were consistent with the results of fluorescent PCR. The results of sequencing of positive samples showed that there was no nonspecific amplification of false positive, and the results showed that, The established GeXP method has the advantages of high throughput, good specificity, high sensitivity, convenience, rapidity, etc. It can be used in clinical differential diagnosis and epidemiological investigation, and has high clinical application value.
【作者单位】：广西兽医研究所广西兽医生物技术重点实验室;
【基金】：广西自然科学基金(2016GXNSFCA380003;2014GXNSFBA118105) 桂科(AB16380003)
【分类号】：S858.23

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