双重荧光定量PCR检测志贺毒素stx1和stx2基因

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102copies/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。

Detection of stx1 and stx2 by duplex quantitative real-time fluorescence polymerase chain reaction

Objective To establish a duplex quantitative real-time PCR method for the detection of stxl and stx2 of Shiga toxin．Methods The PCR primers and TaqMan probes based on the sequences of stxl and stx2 in different bacteria were designed，and the detection system of duplex quantitative real-time PCR was established．Then，the sensitivity，specificity and repeatability of the system were evaluated．The fecal specimens from patients with acute diarrhea were detected by the system．Results The lower detection limit of the duplex quantitative real-time PCR method was 102 copies/mL and the specificity was 100%．The system did not show specific amplifications for the 13 kinds of intestinal pathogenic bacteria．Repeatability study found that the coefficient variation of Ct value was less than 10%，and the positive rate detected by the system was higher than that by the culture method for acute diarrhea fecal samples．Conclusion The established duplex quantitative real-time PCR method could be used to rapidly identify different types of Shiga toxins，and to screen the Shiga toxins in patients with diarrhea．