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双重荧光定量PCR检测志贺毒素stx1和stx2基因

Detection of stx1 and stx2 by duplex quantitative real-time fluorescence polymerase chain reaction
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摘要 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 duplex quantitative real-time PCR method for the detection of stx1 and stx2of Shiga toxin.Methods The PCR primers and TaqMan probes based on the sequences of stx1 and stx2in 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.
作者 陆芸 王若南 谢国良 余斐 郑书发 陈晓 LU Yun, WANG Ruonan, XIE Guoliang, YU Fei, ZHENG Shufa, CHEN Xiao( Department of Clinical Laboratory, The Children's Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang 310052, China)
出处 《中国微生态学杂志》 CAS CSCD 2017年第5期591-593,597共4页 Chinese Journal of Microecology
基金 国家科技重大专项“传染病监测技术平台”项目(2012ZX10004-210)
关键词 志贺毒素 stx1 stx2 实时荧光定量PCR Shiga toxin stx1 stx2 Quantitative real-time fluorescence PCR
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