CAJ | 학술논문

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。
목적：건립일개함확증내대조（IAC）적삼중TaqMan real-time PCR체계，이검측곽란독소기인ctxA화장산독성대장애희균적불내열장독소기인elt。방법침대ctxA、elt화IAC설계인물화탐침，진행령민성화특이성분석，평개삼중반응지간적호상영향。결과해검측체계령민도위ctxA매개반응94고패，elt매개반응79고패，확증효솔분별위94.7%여98.1%。ctxA여elt고패수비례위1∶1～1∶10시，이자균능량호확증；elt혹ctxA적량시IAC적100배이상시，IAC확증수도억제。결론해검측체계구유량호적령민성화특이성，가이용우복사분편중감염병원균적검측，기중내대조검측가이제시분편양본중시부존재PCR억제인자。
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.