Rapid Detection and Typing Method of theEscherichia coliVero Toxin Genes by Fluorescence Polarization

Abstract

Fluorescence polarization technique can specifically detect nucleotide sequences by hybridization without separation procedures. We applied the technique to the detection of the Shiga toxin genes (stx1andstx2) in enterohemorragicEscherichia coli(STEC).
STEC possessesstx1 and/orstx2 and using MK primers, the amplification ofstx1 and/orstx2 can be performed universally, but the band sizes for both genes are almost identical; thus the genotype is hard to determine using electrophoresis. To identify stx genotype, conventional PCR combined with electrophoresis requires a second amplification process with more than 25 temperature cycles using other genotype-specific primer pairs. However, using the newly developed method only a few temperature cycles of the asymmetric PCR using the same MK primers and two different probes forstx1andstx2 were needed. The incubation time for probe hybridization was 10 minutes, and the detection time for fluorescence polarization was within about 1 minute per sample. Thus, we considered that the detection method using fluorescence polarization technique was a rapid and reliable method for detectingstx1and/orstx2.