發(fā)布時(shí)間：2018-04-25 21:00

  本文選題：豬嗜血支原體 + 血液直接PCR�。� 參考：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：目的：建立檢測(cè)M.suis的血液直接PCR方法,篩選具有殺滅M.suis作用的中藥單體。方法：建立血液直接PCR檢測(cè)M.suis的方法；依次采用水、DMSO和無(wú)水乙醇溶解各中藥單體,篩選最佳溶劑；應(yīng)用細(xì)胞病變觀察和MTT法來(lái)測(cè)定各中藥單體在PK-15細(xì)胞培養(yǎng)體系中的最大安全濃度；通過(guò)藥物在M.suis體外維持培養(yǎng)體系中與M.suis直接作用的方式,應(yīng)用血液直接PCR對(duì)藥物作用不同時(shí)間后的培養(yǎng)物進(jìn)行PCR擴(kuò)增,通過(guò)比較電泳條帶灰度值的方法初步篩選出具有抗M.suis作用的有效中藥單體；以血液直接PCR擴(kuò)增片段的克隆為標(biāo)準(zhǔn)品,建立qPCR擴(kuò)增的標(biāo)準(zhǔn)曲線(xiàn),對(duì)有效中藥單體作用后的M.suis培養(yǎng)體系中M.suis的拷貝數(shù)進(jìn)行定量,通過(guò)殺滅率來(lái)評(píng)估各有效中藥單體對(duì)M.suis的殺滅效果。結(jié)果：(1)血液直接PCR可以特異性擴(kuò)增到M.suis 16S rRNA基因中大小為396 bp的片段,而對(duì)PRRSV、PCV2、PPV、PRV、E.coli和S. aureus等病原體沒(méi)有擴(kuò)增出條帶；血液直接PCR的靈敏度與常規(guī)PCR相當(dāng)；血液直接PCR與常規(guī)PCR對(duì)樣品檢測(cè)無(wú)顯著差異(p0.05)。(2)篩選了各中藥單體的最佳溶劑；確定了各中藥單體對(duì)PK-15細(xì)胞的安全濃度。(3)應(yīng)用建立的血液直接PCR初步篩選出丹參酮Ⅱ A磺酸鈉、甘草酸二鉀、秦皮甲素、黃芩苷和肉桂酸5種具有體外抗M.suis作用的中藥單體。(4)通過(guò)qPCR檢測(cè)5種中藥單體對(duì)M.suis的殺滅效果,隨著藥物作用時(shí)間的延長(zhǎng),5種中藥單體對(duì)M.suis的殺滅率均顯著升高。其中,丹參酮ⅡA磺酸鈉、甘草酸二鉀和秦皮甲素在藥物作用18h后,殺滅率分別達(dá)到了76.9%、90.6%和79.2%。結(jié)論：1.建立了檢測(cè)M.suis感染的血液直接PCR方法。2篩選出了甘草酸二鉀、丹參酮ⅡA磺酸鈉、秦皮甲素、黃芩苷和肉桂酸5種具有體外抗M.suis作用的中藥單體。其中,丹參酮ⅡA磺酸鈉、甘草酸二鉀和秦皮甲素對(duì)M.suis的殺滅效果較好,在藥物作用18 h后,對(duì)M.suis的殺滅率均達(dá)到了70%以上,是理想的抗M.suis藥物。
[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，

本文編號(hào)：1802943