【摘要】：目的：通過(guò)使用普通兩步法RT-PCR與Real-time PCR檢測(cè)糞腸球菌毒力因子gelE的表達(dá)，評(píng)價(jià)Real-time PCR技術(shù)測(cè)定糞腸球菌mRNA表達(dá)水平的價(jià)值。 方法：培養(yǎng)糞腸球菌國(guó)際標(biāo)準(zhǔn)株ATCC29212，提取細(xì)菌基因組總RNA,測(cè)定基因組總RNA濃度，將基因組總RNA按照1:10-1:107進(jìn)行梯度稀釋,將RNA逆轉(zhuǎn)錄cDNA,采用Real-time PCR及RT-PCR檢測(cè)糞腸球菌gelE基因表達(dá)水平的差異。 結(jié)果：RT-PCR檢測(cè)可見(jiàn)有陽(yáng)性擴(kuò)增條帶的最大稀釋度為1：103，，基因表達(dá)敏感度為550pg/ul。Real-time PCR在稀釋為1：105仍出現(xiàn)陽(yáng)性擴(kuò)增曲線，其基因表達(dá)敏感度為5.5pg/ul, Real-time PCR在測(cè)定糞腸球菌毒力因子gelE敏感度下限是常規(guī)RT-PCR的100倍。 結(jié)論：1RT-PCR及Real-time PCR都可作為檢測(cè)糞腸球菌mRNA定量分析的方法。 2檢測(cè)糞腸球菌毒力因子gelE的方法, Real-time PCR法的靈敏度較普通的兩步法RT-PCR高100倍。
[Abstract]:Aim: to detect the expression of virulence factor gelE (gelE) of Enterococcus faecalis by RT-PCR and Real-time PCR, and to evaluate the value of Real-time PCR technique in detecting mRNA expression of Enterococcus faecalis. Methods: the genomic total RNA, of Enterococcus faecalis was extracted from the international standard strain of Enterococcus faecalis. The total RNA concentration was determined. The total genomic RNA was diluted by gradient dilution according to 1: 10-1: 107, and the RNA reverse transcription cDNA, was performed. Real-time PCR and RT-PCR were used to detect the expression of gelE gene in Enterococcus faecalis. Results: RT-PCR showed that the maximum dilution of positive amplified bands was 1: 103, the sensitivity of gene expression was that 550pg/ul.Real-time PCR still appeared positive amplification curve at dilution of 1: 105, the sensitivity of gene expression was 5.5 PG / ul. The lower sensitivity of Real-time PCR in determining enterococcus faecalis virulence factor gelE was 100 times higher than that of conventional RT-PCR. Conclusion: both 1RT-PCR and Real-time PCR can be used to detect mRNA quantitative analysis of Enterococcus faecalis. 2 the sensitivity of Real-time PCR method for detecting enterococcus faecalis virulence factor gelE was 100 times higher than that of RT-PCR.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R780.2;R3416

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