A multiplex polymerase chain reaction （PCR） was developed to detect three pathogenic genes of enteropathogenic, enterotocigenic and enteroinvasive Escherichia coli In this study three different sets of oligonucleoti...A multiplex polymerase chain reaction （PCR） was developed to detect three pathogenic genes of enteropathogenic, enterotocigenic and enteroinvasive Escherichia coli In this study three different sets of oligonucleotide primer were simultaneously used, and in this way, specific fragments of 880, 600, 150 bp for EPEC eaeA, EIEC ipaH and ETEC ST genes were amplified, respectively. The best condition of the multiplex PCR was： after an initial heat denaturation step at 95℃for 5 min, followed by 30 cycles of denaturation at 94 ℃ for 40 s, primer annealing at 51.3℃ for 40 s and extension at 72 ℃ for 1 min, final extension at 72 ℃ for 10 min. The detection limit of the eaeA, ipaH and ST primers was 38.7423, 3.60519, 29.9448 ng·mL^-1 （4.3×10^4, 1.5×10^3, 2.6×10^4 CFU·mL^-1）, respectively. It may be a good way for the detection and identification of Diarrhea-causing E. coli.展开更多
文摘【目的】本研究探讨了云南撒坝猪EPEC HPI毒力岛的摄铁功能与致病性之间的关系。【方法】本试验通过摄铁实验对5株肠致病性E.coli(A-E)和CVCC1565铁载体的合成和在不同2,2’-联吡啶含量下的最低生长抑制浓度及对数期生长速率进行了测定,同时利用已构建的E.coli irp2基因缺失株和亲本株进行对比,检测HMWP2蛋白的表达,并通过半数致死量实验比较两者之间的毒力。【结果】除E.coli(D)外,其余菌株的2,2’-联吡啶最低生长抑制浓度为0.6 m M;亲本株能够表达HMWP2蛋白,irp2缺失株不表达该蛋白;CAS固体培养基中菌株能产生橘黄色的透明晕圈;当2,2’-联吡啶浓度高于0.2 m M时,亲本株的生长率高于irp2缺失株;亲本株的LD50(8.73×10~7cfu/m L)低于缺失株(9.95×10~7cfu/m L)。【结论】云南撒坝猪EPEC HPI铁摄取功能和致病性之间存在着一定关系。
基金Key Item of National Technology Research Project (2002BA518A06)Heilongjiang Province Department Fund (10541021)
文摘A multiplex polymerase chain reaction （PCR） was developed to detect three pathogenic genes of enteropathogenic, enterotocigenic and enteroinvasive Escherichia coli In this study three different sets of oligonucleotide primer were simultaneously used, and in this way, specific fragments of 880, 600, 150 bp for EPEC eaeA, EIEC ipaH and ETEC ST genes were amplified, respectively. The best condition of the multiplex PCR was： after an initial heat denaturation step at 95℃for 5 min, followed by 30 cycles of denaturation at 94 ℃ for 40 s, primer annealing at 51.3℃ for 40 s and extension at 72 ℃ for 1 min, final extension at 72 ℃ for 10 min. The detection limit of the eaeA, ipaH and ST primers was 38.7423, 3.60519, 29.9448 ng·mL^-1 （4.3×10^4, 1.5×10^3, 2.6×10^4 CFU·mL^-1）, respectively. It may be a good way for the detection and identification of Diarrhea-causing E. coli.
