Abstract
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 μl 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°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.
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Kim, IW., Kang, MH., Kwon, SH. et al. Rapid detection of virulence stx2 gene of Enterohemorrhagic Escherichia coli using two-step ultra-rapid real-time PCR. Biotechnol Lett 32, 681–688 (2010). https://doi.org/10.1007/s10529-010-0205-0
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DOI: https://doi.org/10.1007/s10529-010-0205-0