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不同实验方法检测粪肠球菌毒力因子gelE敏感度差异

发布时间:2019-02-11 21:20
【摘要】:目的:通过使用普通两步法RT-PCR与Real-time PCR检测粪肠球菌毒力因子gelE的表达,评价Real-time PCR技术测定粪肠球菌mRNA表达水平的价值。 方法:培养粪肠球菌国际标准株ATCC29212,提取细菌基因组总RNA,测定基因组总RNA浓度,将基因组总RNA按照1:10-1:107进行梯度稀释,将RNA逆转录cDNA,采用Real-time PCR及RT-PCR检测粪肠球菌gelE基因表达水平的差异。 结果:RT-PCR检测可见有阳性扩增条带的最大稀释度为1:103,,基因表达敏感度为550pg/ul。Real-time PCR在稀释为1:105仍出现阳性扩增曲线,其基因表达敏感度为5.5pg/ul, Real-time PCR在测定粪肠球菌毒力因子gelE敏感度下限是常规RT-PCR的100倍。 结论:1RT-PCR及Real-time PCR都可作为检测粪肠球菌mRNA定量分析的方法。 2检测粪肠球菌毒力因子gelE的方法, Real-time PCR法的灵敏度较普通的两步法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.
【学位授予单位】:大连医科大学
【学位级别】:硕士
【学位授予年份】:2012
【分类号】:R780.2;R3416

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