猪嗜血支原体血液直接PCR方法的建立及抗猪嗜血支原体中药单体的体外筛选

发布时间：2018-04-25 21:00

  本文选题：猪嗜血支原体 + 血液直接PCR　； 参考：《山西农业大学》2015年硕士论文

【摘要】：目的：建立检测M.suis的血液直接PCR方法,筛选具有杀灭M.suis作用的中药单体。方法：建立血液直接PCR检测M.suis的方法；依次采用水、DMSO和无水乙醇溶解各中药单体,筛选最佳溶剂；应用细胞病变观察和MTT法来测定各中药单体在PK-15细胞培养体系中的最大安全浓度；通过药物在M.suis体外维持培养体系中与M.suis直接作用的方式,应用血液直接PCR对药物作用不同时间后的培养物进行PCR扩增,通过比较电泳条带灰度值的方法初步筛选出具有抗M.suis作用的有效中药单体；以血液直接PCR扩增片段的克隆为标准品,建立qPCR扩增的标准曲线,对有效中药单体作用后的M.suis培养体系中M.suis的拷贝数进行定量,通过杀灭率来评估各有效中药单体对M.suis的杀灭效果。结果：(1)血液直接PCR可以特异性扩增到M.suis 16S rRNA基因中大小为396 bp的片段,而对PRRSV、PCV2、PPV、PRV、E.coli和S. aureus等病原体没有扩增出条带；血液直接PCR的灵敏度与常规PCR相当；血液直接PCR与常规PCR对样品检测无显著差异(p0.05)。(2)筛选了各中药单体的最佳溶剂；确定了各中药单体对PK-15细胞的安全浓度。(3)应用建立的血液直接PCR初步筛选出丹参酮Ⅱ A磺酸钠、甘草酸二钾、秦皮甲素、黄芩苷和肉桂酸5种具有体外抗M.suis作用的中药单体。(4)通过qPCR检测5种中药单体对M.suis的杀灭效果,随着药物作用时间的延长,5种中药单体对M.suis的杀灭率均显著升高。其中,丹参酮ⅡA磺酸钠、甘草酸二钾和秦皮甲素在药物作用18h后,杀灭率分别达到了76.9%、90.6%和79.2%。结论：1.建立了检测M.suis感染的血液直接PCR方法。2筛选出了甘草酸二钾、丹参酮ⅡA磺酸钠、秦皮甲素、黄芩苷和肉桂酸5种具有体外抗M.suis作用的中药单体。其中,丹参酮ⅡA磺酸钠、甘草酸二钾和秦皮甲素对M.suis的杀灭效果较好,在药物作用18 h后,对M.suis的杀灭率均达到了70%以上,是理想的抗M.suis药物。
[Abstract]:Objective: to establish a direct PCR method for the detection of M.suis and to screen out Chinese traditional medicine monomers which can kill M.suis. Methods: to establish a method for the detection of M.suis by direct PCR in blood, and then dissolve the Chinese traditional medicine monomer with water DMSO and anhydrous ethanol in order to select the best solvent. The maximum safe concentration of Chinese traditional medicine monomer in PK-15 cell culture system was determined by cytopathic observation and MTT method, and the direct interaction with M.suis in M.suis culture system was maintained by drugs in vitro. Blood direct PCR was used to amplify the culture of different time after the drug action. By comparing the gray value of the electrophoresis band, the effective Chinese medicine monomer with anti- effect was preliminarily selected. The standard curve of qPCR amplification was established by using the clone of blood direct PCR amplification fragment. The copy number of M.suis in M.suis culture system after the effective Chinese medicine monomer was acted on was quantified. The killing rate was used to evaluate the killing effect of each effective Chinese medicine monomer on M.suis. Results the specific fragment of M.suis 16s rRNA gene was amplified by direct PCR in blood, but not by E. coli and S. aureus in PRRSV-PCV2, and the sensitivity of direct PCR in blood was similar to that of conventional PCR. The best solvent for each Chinese medicine monomer was screened by direct PCR and routine PCR. The safe concentration of PK-15 cells was determined by the method of direct PCR. The sodium tanshinone 鈪，

本文编号：1802943