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
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.
