nklin大肠杆菌ompT基因的相对表达定量PCR方法的建立

Detection of Relative Expression of ompT Gene in Escherichia coli by SYBR Green I RT-PCR

目的建立ompT基因表达水平解析的定量方法。方法利用嵌合荧光（SYBR GreenI）标记，以编码甘油醛3-磷酸脱氢酶的看家基因gapA为内参基因，建立用于ompT基因表达水平解析的两步法实时定量反转录PCR。通过大肠杆菌的鸡体内感染模型，获取感染菌体，利用该定量PCR对感染菌体中ompT基因在感染鸡体内的表达水平进行了解析。结果成功建立了ompT基因表达水平解析的定量PCR。定量PCR结果显示，相对于体外培养，APECE058株ompT在鸡体内的表达上调了6．69倍。结论建立的ompT基因定量PCR方法稳定、可靠，可用于ompT表达水平的解析。

Objective To establish a method to relatively quantify the expression of ompT and the gene of APEC strain E058 in vivo. Methods Two-step real time quantitative RT-PCRs （qRT-PCR） of ompT and gapA were developed based on SYBR Green I, respectively. In these qRT-PCRs, ompT was identified as the target gene and gapA as internal reference, and two standard curves were established using a series dilution of cDNA synthesized from the RNA of APEC E058 grown statically to exponential phase in rich medium. Results qRT-PCR of ompT was established. In chicken challenge model, the expressions of ompT in APEC E058 were up-regulated 6.69-fold compared to that of APEC E058 grown statically to exponential phase in rich medium. Conclusion The established qRT-PCR was reliable for the expression analysis of ompT.