金黄色葡萄球菌基因表达的DNA扣除法Real-time RT PCR相对定量分析

Real-time RT PCR with DNA subtraction for relative quantification of gene expression in Staphylococcus aureus

【目的】金黄色葡萄球菌作为一种分布广泛的致病微生物和研究革兰氏阳性菌遗传背景的模式菌株,利用real-time RT PCR对相关毒素及调控基因进行表达定量分析,在生物、医学、食品检测等领域具有较大研究价值。【方法】对制备好的反转录(RT,含有cDNA和DNA)和非反转录(RT—,仅含DNA)样品进行Real-time PCR检测,根据经典(1+E)—△△Ct相对定量算法并结合PCR效率公式建立一种基因表达相对定量分析的DNA扣除法,将得到的Ct值转换为各样品含量,从RT样品中扣除RT—样品的量,无需DNaseⅠ酶解处理就可以去除DNA的影响,RT—样品的检测结果还可同时作为稳定的DNA内参。【结果】采用以上方法分析金黄色葡萄球菌肠毒素A基因(sea)、16S rRNA和RNAⅢ的表达情况,在含有葡萄糖的NB培养基中sea的相对转录水平随着葡萄糖浓度的增大而升高,RNAⅢ的相对转录水平随葡萄糖浓度的变化而产生小幅度的波动,16S rRNA在菌体生长初期时的表达量较为稳定;与绝对定量法比较,结果差异较小(均小于15%),且差异不显著(p>0.05)。【结论】这种基于DNA扣除法的Real-time RT PCR相对定量方法可以有效的对金黄色葡萄球菌的基因表达进行分析。

[Objective] We evaluated relative quantification by real-time RT PCR of a target gene transcription. [Methods] On the basis of （I＋E）^-△△Ct mathematical model and the E=10^[-l/slopel-1 equation, the detected Ct data of the real-time RT PCR was analyzed by the new DNA subtraction assay. DNA was used as standard for the initial amount of bacteria. RT and RT^- samples for real-time PCR detection were prepared to quantify the DNA that simultaneously existed with RNA isolated from the bacteria samples. The detected quantitative data were subtracted from total nucleic acid simultaneously contained RNA and DNA. Enzymatic digestion with DNase I was not included in this protocol. [Results] The gene expression of staphylococcal enterotoxin A （sea）, 16S rRNA and RNAⅢ of Staphylococcus aureus were detected. These two different analysis methods, DNA subtraction method and absolute quantitative method, led to similar results （p〉0.05）. [Conclusion] This is a time-saving and efficient method. Additionally, for further studies it would be conceivable to extend the detection of genes expression from S. aureus to other prokaryote.