| 李杰,阚飙,张京云.含扩增内对照的霍乱毒素基因ctx和不耐热肠毒素基因elt三重real-time PCR检测体系的建立[J].中华流行病学杂志,2014,35(6):720-723 |
| 含扩增内对照的霍乱毒素基因ctx和不耐热肠毒素基因elt三重real-time PCR检测体系的建立 |
| Establishment of a triplex real-time PCR for the detection of cholera toxin gene ctx and heat labile enterotoxin gene elt |
| 收稿日期:2014-02-10 出版日期:2014-09-02 |
| DOI： |
| 中文关键词: 霍乱毒素 不耐热肠毒素 实时荧光定量聚合酶链式反应 |
| 英文关键词: Cholera toxin Heat-labile enterotoxin Real-time PCR |
| 基金项目:国家科技重大专项（2013ZX-100040101） |
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| 中文摘要: |
| 目的 建立一个含扩增内对照（IAC）的三重TaqMan real-time PCR体系，以检测霍乱毒素基因ctxA 和肠产毒性大肠埃希菌的不耐热肠毒素基因elt。方法 针对ctxA、elt 和IAC设计引物和探针，进行灵敏性和特异性分析，评价三重反应之间的互相影响。结果 该检测体系灵敏度为ctxA 每个反应94 拷贝，elt 每个反应79 拷贝，扩增效率分别为94.7%与98.1%。ctxA 与elt 拷贝数比例为1 ∶ 1～1 ∶ 10 时，二者均能良好扩增;elt 或ctxA 的量是IAC的100 倍以上时，IAC扩增受到抑制。结论 该检测体系具有良好的灵敏性和特异性，可以用于腹泻粪便中感染病原菌的检测，其中内对照检测可以提示粪便样本中是否存在PCR抑制因子。 |
| 英文摘要: |
| Objective To establish a triplex TaqMan real-time PCR system containing internal amplification control（IAC）to detect cholera toxin gene ctxA and enterotoxigenic Escherichia coli（ETEC）heat-labile enterotoxin gene elt. Methods Primers and probes were designed based on the sequences of ctxA，elt and IAC. Both sensitivity and specificity were analyzed and interactions between different reactions were evaluated. Results This system showed that the sensitivity of ctxA was 94 copies/reaction while the elt 79 copies/reaction and the amplification efficiency were 94.7% and 98.1%，respectively. Under the ratio of copy numbers on gene ctxA to elt as between 1 ∶ 1-1 ∶ 10， when both targets were detected，with impact was less on each other. However，when the amount of elt or ctxA was 100 times of IAC，the amplification of IAC was significantly inhibited. Conclusion This system showed both satisfactory sensitivity and specificity，thus could be used to detect pathogenic bacteria in diarrhea stools. The detection of IAC could prompt the presence of PCR inhibitors in samples being tested. |
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