实时荧光定量PCR检测感染小鼠肠道内容物的LT肠毒素基因

构建LT-PMD19质粒标准品,进行SYBR Green荧光定量PCR检测产肠毒素大肠杆菌(ETEC)LT基因,以确定该方法的特异性和灵敏度,绘制标准曲线;通过腹腔注射0.2mL ETEC菌液建立小鼠腹泻模型,分别于感染后4h、15h、2d、7d和14d各处死2只,通过建立的荧光定量PCR方法对小肠液中的ETEC的LT基因进行定量检测。研究结果显示,用建立的荧光定量PCR检测LT基因无非特异性扩增,标准曲线呈良好的线性关系(R2=0.999),标准品的熔解曲线均呈单峰,灵敏度为8.18×103拷贝数/μL;小鼠感染4h后LT毒素含量最高,剖检可见小肠肿大,充满黄绿色的内容物,感染后7d、14d均未检测出LT毒素,肠道未见明显病变,表明LT肠毒素在肠道内的生成规律与肠道病变损伤呈正相关。

The Content of LT Gene in Intestine of Mice by Using Real time Quantitative PCR

Constructing LT-PMD19 plasmid was served as standard product to determine the specificity and sensitivity of SYBR Green fluorescence quantitative PCR for detection of heat-labile enterotoxin(LT) produced by the enterotoxigenic Escherichia coli(ETEC) and to prepare a standard curve.To establish the mouse model,0.2 mL ETEC fluid was injected into the enterocoelia.The mice were slaughtered at 4 h,15 h,2 d,7 d and 14 d after infection,respectively.The intestinal juice was detected by real-time PCR.The results showed that the designed primer was specific for q-PCR detection of LT and melting curves were single peak.Standard curve had a good linear relationship(R2=0.999) and the sensitive degree was 8.18×103 copies/μL.The LT toxin content in infected mouse was peak on 4 h post infection.Postmortem examination showed that the small intestine was swelling and full of yellow-green contents.LT toxin was not detected at 7 d and 14 d post infection,the small intestine has not obvious pathological changes,which was a positive correlation expressed between the content of LT toxin in intestine and the level of intestinal damage.