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目的建立一种双重荧光定量PCR检测志贺毒素stx1和stx2基因的方法。方法根据不同细菌来源的stx1和stx2序列,设计PCR引物和TaqMan探针,建立双重实时荧光定量PCR检测体系,进行灵敏度、特异性和重复性评价,并对腹泻患者粪便样本进行检测分析。结果双重实时荧光定量PCR检测含志贺毒素基因重组质粒的最低检测下限为102 copies/mL;该法对12种常见肠道病原菌均无特异性扩增,对不同浓度的标准质粒检测重复性高,Ct值变异系数均小于10%;对急性腹泻粪便标本的检测阳性率高于细菌分离培养。结论建立的双重实时荧光定量PCR可作为不同细菌来源的志贺毒素基因的快速鉴定方法,亦可用于人感染性腹泻标本的快速筛查。
Objective To establish a double fluorescence quantitative PCR method for detecting Shiga toxin stx1 and stx2 genes. Methods Based on the stx1 and stx2 sequences of different bacterial sources, PCR primers and TaqMan probes were designed and used to establish double real-time fluorescence quantitative PCR system to evaluate the sensitivity, specificity and repeatability. The stool samples of diarrhea patients were detected and analyzed. Results The detection limit of recombinant plasmid containing Shiga toxin gene by double real-time PCR was 102 copies / mL. No specific amplification was found for the 12 common gut pathogens, and the repeatability of the standard plasmids with different concentrations was high , The coefficient of variation of Ct value was less than 10%; the detection rate of stool specimens for acute diarrhea was higher than that of bacterial isolation and culture. Conclusion The established double real-time fluorescence quantitative PCR can be used as a rapid identification method for Shiga toxin genes of different bacterial sources, and can also be used for rapid screening of human infectious diarrhea specimens.