Rapid detection of virulence stx2 gene of Enterohemorrhagic Escherichia coli using two-step ultra-rapid real-time PCR.
A rapid detection method for Enterohemorrhagic Escherichia coli (EHEC), which has the virulent stx2 gene, was developed using a two-step, ultra-rapid real-time (URRT) PCR. URRT PCR was designed to detect the stx2 gene using a microchip-based, real-time PCR system, GenSpector TMC-1000, which only has a 6 microl total reaction volume with an extremely short denaturation step and combined annealing/extension step (1 and 3 s, respectively) for each cycle. Specific primers for the stx2 gene were designed to amplify a 100 bp region known for genetic stability among the various EHEC strains. Using the URRT PCR method, stx2 gene could be detected in 7 min 8 s including melting point (Tm) analysis. The detection limit for the stx2 gene for URRT-PCR was estimated to be 3 c.f.u./PCR with the amplification product having a consistent Tm of 85.2 +/- 0.4 degrees C. This method was tested for the various applications relevant to the different EHEC strains and was useful for the rapid detection of stx2-carrying EHEC strains.
Kim
Il-Wook
IW
Department of Life Science, College of Natural Science, Kyonggi University, Suwon, 443-760, Korea.
Kang
Min-Hee
MH
Kwon
Soon-Hwan
SH
Cho
Seung-Hak
SH
Yoo
Byung-Sun
BS
Han
Sang-Hoon
SH
Yoon
Byoung-Su
BS
D001431:Bacteriological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D054324:Enterohemorrhagic Escherichia coli / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022641:Shiga Toxin 2 / Q000096:biosynthesis* / Q000235:genetics; D013997:Time Factors; D044366:Transition Temperature; D037521:Virulence Factors / Q000096:biosynthesis* / Q000235:genetics
D017931:DNA Primers; D029968:Escherichia coli Proteins; D022641:Shiga Toxin 2; D037521:Virulence Factors