CAJ | 학술논문

目的应用两组三重PCR方法检测非O157:H7型STEC的6种毒力基因stx1、stx2、eae、hly、etpD、katP.方法对多重PCR方法进行改进优化,引入SYBR Green Ⅰ荧光定量PCR熔解曲线分析的方法提高工作效率,并结合血清学分析的方法判定结果.建立检测产志贺毒素大肠埃希菌毒力基因的多重PCR方法,简化其分子危害性评估工作,提高检测的准确性与评估效率.结果用模拟样品评估该方法,检测下限分别为:组1 stx1基因10 ng/ml、stx2基因120 ng/ml、eaeP基因110ng/ml;组二etpD基因165 ng/ml、katP基因85 ng/ml、hly基因15 ng/ml.其特异性和灵敏度与单一基因PCR扩增结果基本一致；而运用该方法对210例腹泻患者样品进行检测应用,得到13株非O157STEC菌株,其毒力基因为stx1基因均阳性,eae基因伴随存在,而hly、etpD、katP毒力基因呈散发性分布.结论本研究建立的方法能够快速有效地进行非O157 STEC的毒力基因的检出和排查,既提高了检测效率,又可作为评估分子危害性的检查方法.
목적 응용량조삼중PCR방법검측비O157:H7형STEC적6충독력기인stx1、stx2、eae、hly、etpD、katP.방법 대다중PCR방법진행개진우화,인입SYBR Green Ⅰ형광정량PCR용해곡선분석적방법제고공작효솔,병결합혈청학분석적방법판정결과.건립검측산지하독소대장애희균독력기인적다중PCR방법,간화기분자위해성평고공작,제고검측적준학성여평고효솔.결과 용모의양품평고해방법,검측하한분별위:조1 stx1기인10 ng/ml、stx2기인120 ng/ml、eaeP기인110ng/ml;조이etpD기인165 ng/ml、katP기인85 ng/ml、hly기인15 ng/ml.기특이성화령민도여단일기인PCR확증결과기본일치；이운용해방법대210례복사환자양품진행검측응용,득도13주비O157STEC균주,기독력기인위stx1기인균양성,eae기인반수존재,이hly、etpD、katP독력기인정산발성분포.결론 본연구건립적방법능구쾌속유효지진행비O157 STEC적독력기인적검출화배사,기제고료검측효솔,우가작위평고분자위해성적검사방법.
Objective Traditional detection approaches for non-O157 STEC are both time and labour consuming in diseases surveillance.Virulence genes detection based on multiplex PCR could not only improve the detection efficiency but also increase the accuracy.Methods Six virulence genes of nonO157:H7 (stx1,stx2,eae,hly,etpD,katP6) were detected by two groups of trebling PCRs.The multiplex PCRs were optimized by melting curve analysis in SYBR Green Ⅰ real-time PCR.Testing result of multiplex PCR was consistent with serological testing.Results The sensitivity limits of the multiplex PCR for stx1,stx2,eaeP,etpD,katP,and hly were 10 ng/ml,120 ng/ml,110 ng/ml,165 ng/ml,85 ng/ml,and 15 ng/ml,respectively,which is similar with that of single PCR.When the multiplex PCR was applied in 120 adults and 90 children diarrhea samples detection,13 cases were detected for non-O157 positive.Conclusion The method we established can be used for non-O157 STEC virulence genes detection and screening with high efficiency and accuracy.