發(fā)布時(shí)間：2018-05-31 21:20

  本文選題：新生隱球菌 + 活力　； 參考：《第二軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：新生隱球菌和格特隱球菌是重要的病原真菌，常常引起致命的腦膜炎；后者治療時(shí)間長(zhǎng)，，復(fù)發(fā)率較高。目前在隱球菌腦膜炎及其它系統(tǒng)感染的治療中困擾臨床醫(yī)生的一個(gè)重要問(wèn)題，就是由于無(wú)法在治療過(guò)程中對(duì)隱球菌的活力做出有效的準(zhǔn)確判斷，從而對(duì)療程的判定缺乏客觀的時(shí)間標(biāo)準(zhǔn)。檢測(cè)rRNA和rRNA基因判斷微生物活力，尤其是對(duì)于判斷微生物在“活而不可培養(yǎng)”狀態(tài)下的活力有很強(qiáng)的特異性和敏感性。通過(guò)檢測(cè)rRNA判斷微生物活性在麻風(fēng)桿菌、沙門氏菌、釀酒酵母、念珠菌等都顯示良好的作用，而對(duì)于新生隱球菌和格特隱球菌的研究卻未見報(bào)道，本課題研究目的是通過(guò)檢測(cè)存活和熱滅活的隱球菌rRNA和rRNA基因的特異性片段，探尋判定新生隱球菌活力的特異性rRNA，為后續(xù)實(shí)驗(yàn)研究奠定基礎(chǔ)。 從隱球菌專業(yè)中心實(shí)驗(yàn)室取新生隱球菌標(biāo)準(zhǔn)株（B3501）接種到固體沙氏培養(yǎng)基活化三天。三天后轉(zhuǎn)種一整環(huán)菌體于YEPD液體培養(yǎng)基，振蕩混勻后置于振蕩培養(yǎng)箱（200r/min，30℃）過(guò)夜。振蕩培養(yǎng)過(guò)夜后，菌懸液理論濃度應(yīng)該在106-107cfu/ml,以保證后續(xù)實(shí)驗(yàn)中能提取足夠的總RNA;將其中100ml菌懸液置于燒瓶A中，定義為對(duì)照組（活菌組），將剩余100ml菌懸液置于燒瓶B中，并用高壓鍋（121℃,15min）熱力滅活，定義為實(shí)驗(yàn)組（死菌組）。將對(duì)照組和實(shí)驗(yàn)組分別用離心機(jī)（5000rpm）提取菌體，加入Trizol后，放置室溫，提取總RNA后使用試劑盒進(jìn)行逆轉(zhuǎn)錄，合成為cDNA模板，而后以cDNA為模板，進(jìn)行Real-time PCR實(shí)驗(yàn)。數(shù)據(jù)采用SPSS18.0統(tǒng)計(jì)軟件包進(jìn)行分析，結(jié)果以均數(shù)±標(biāo)準(zhǔn)差（x±s）表示；計(jì)量資料兩組之間采取獨(dú)立樣本t檢驗(yàn)，以P＜0.05為具有統(tǒng)計(jì)學(xué)差異，P＜0.01為具有顯著統(tǒng)計(jì)學(xué)差異。 1.5%瓊脂糖凝膠電泳檢測(cè)顯示總RNA18s，28s條帶清晰，并且28s的量為18s的2倍，表明所得RNA無(wú)降解，逆轉(zhuǎn)錄后合成cDNA，最后通過(guò)RT-PCR方法檢測(cè)5s，5.8s，18s，28s四個(gè)小亞基，死菌和活菌之間表達(dá)量均有差異性，CT值差異從大到小依次為：18s，5.8s，28s，5s，且均有統(tǒng)計(jì)學(xué)意義（P＜0.001）。以CT值差異最小的5s小亞基作為內(nèi)參，將Ct值轉(zhuǎn)化為△Ct值，結(jié)果顯示18s活菌與死菌的表達(dá)量相差45倍，5.8s相差14倍，通過(guò)本課題的初步探討和研究得出了以下結(jié)論。 采用RT-PCR方法檢測(cè)新生隱球菌四個(gè)小亞基的表達(dá)量可以對(duì)新生隱球菌的活力做出初步判斷，尤其是18s和5.8s小亞基，可作為關(guān)鍵亞基，進(jìn)行檢測(cè)做出判斷。
[Abstract]:Cryptococcus neoformans and Cryptococcus gertsii are important pathogenic fungi, which often cause fatal meningitis; the latter has a long treatment time and a high recurrence rate. One of the most important problems that currently plague clinicians in the treatment of Cryptococcus meningitis and other systemic infections is the inability to make an effective and accurate judgment of Cryptococcus activity during the course of treatment. Therefore, the judgment of the course of treatment lacks the objective time standard. The detection of rRNA and rRNA genes for the determination of microbial vitality, especially for the determination of microbial activity in the "live but unculturable" state has a strong specificity and sensitivity. The detection of rRNA showed that microbial activity had a good effect on leprosy, salmonella, Saccharomyces cerevisiae and candida, but the studies on Cryptococcus neoformans and Cryptococcus Gertrudii were not reported. The purpose of this study was to detect the specific fragments of the rRNA and rRNA genes of Cryptococcus neoformans and to find out the specific rRNAs to determine the activity of Cryptococcus neoformans and to lay a foundation for further experimental research. The standard Cryptococcus neoformans strain B3501 was inoculated into solid Salmonella medium for three days. After three days, a whole cell was transferred to YEPD liquid medium, and then mixed in a shaking incubator at 200 r / min ~ (30 鈩

本文編號(hào)：1961320