摘要
布鲁氏菌病是一种危害严重的人兽共患病,在世界范围内广泛流行。我国布鲁氏菌病主要的诊断方法为细菌分离培养和血清学检测(虎红平板凝集和试管凝集试验),但满足不了快速检测的要求。本论文开展布鲁氏菌快速检测方法的研究,从血清学抗体筛查→阳性样品的病原学检测→病原的分型鉴定,建立系列的检测方法,为布鲁氏菌病净化检疫,提供快速有效地手段。
1.建立胶体金试纸检测方法,适用于现场检测。根据双抗原夹心法测抗体的原理,用葡萄球菌蛋白A和布鲁氏菌重组蛋白bp26抗原建立检测布鲁氏菌抗体的胶体金免疫层析试纸条。特异性试验结果表明,阳性和阴性血清检测结果正确,说明该方法特异性较好。试纸条保质期为4℃保存6个月。
2.建立荧光定量PCR检测方法,适用于检测病原。以布鲁氏菌属OMP10保守片段为靶基因,设计特异性引物和Taqman荧光探针。将OMP10基因片段克隆到pMD19-T载体,提取质粒,制备标准品及标准曲线。特异性分析结果显示,对照菌株均未出现荧光信号,说明该方法特异性较好；灵敏性分析表明,该方法最低检出数量级为为10-4ng/μL:标准质粒的稳定性试验结果表明,在不同检测时间、不同反应管之间的域值的变化很小,组内组间变异系数均小于2%。
3.建立布鲁氏菌分型多重PCR方法,适用于病原进行分型。根据布鲁氏菌基因组多拷贝插入元件IS711及其下游多态位点,设计布鲁氏菌牛、羊、猪种分型引物,优化PCR反应体系和条件,建立布鲁氏菌分型的多重PCR方法。特异性分析结果显示,对照菌株未见扩增,说明该方法特异性较好。灵敏性分析表明,其最低检出数量级为10-2ng/μL.
Brucellosis is one kind of amphixenosis, which is severely impaired in the world. The detection methods in my Country are bacteriology and serology (RBT and SAT). But. these methods have many drawbacks. It is necessary to establish new methods to improve detection level. This study established three methods to lay a foundation for Brucellosis control. To meet the demand of the quarantine purification of brucellosis, the papers study on the rapid detection method of brucellosis, the serological typing identification of pathogen detection antibody screening→positive samples→pathogens.
1. Established colloidal gold immunochromatography assay(GICA) for the field detection brucella.According to double antigen sandwich detection antibody.we used SPA and bp26to set up the colloidal gold immunity chromatography test paper.The specific test showed the detection result of control serum were Correct.The sensitivity of test reached the level of SAT.The shelf life was6months in4℃.
2. Established fluorescence quantitative PCR(FQ-PCR)for the quantitative detection brucella. According to the consensus sequence of brucella genome OMP10, we devised specific primer and Taqman fluorescent probe.We had cloned the gene part OMP10to carrier PMD19-T,extracted plasmid and prepared the standard substance and the standard curve.The result of specific testing showed that there was not fluorescence signal in compare bacterias.By the analysis of sensitivity,we heaved known that the lowest detection limit was10-4ng/μL.The threshold change was little in different test time and different reaction tubes in stability experiment and the coefficient of variability was less than2%.
3.Established multiplex PCR for detection and classification of Brucella.According to multicopy insertion element IS711and its polymorphic site on lower reaches,we had devised primers of detection brucella and classification cow, sheep and pig brucella. The multiple PCR reaction system and condition had been optimized.The multiple PCR which was to detect and classificate the brucella of cow、sheep and pig had been set up. By detecting the comparing bacterial strain.there were not specificity amplification.This method was specificity.By analyzing the sensitivity.the method of multiple PCR could test the10-2ng/μL.
引文
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