[Rapid detection of the Escherichia cori verotoxin gene using fluorescence polarization].
The effects of NaCl concentration, temperature and base-pair mismatches on the hybridization of two complementary single-stranded DNA 24-mers were investigated using a fluorescence polarization method. Over a temperature range of 46 degrees C to 56 degrees C in 0.8 M NaCl it was found that hybridization was essentially complete in under 10 minutes and that rapid in vitro determination of the target DNA was possible when the sample DNA had three or less base-pair mismatches in the 24-mer sequence. Polarization measurements on positive and negative samples showed excellent agreement with results obtained from electrophoresis. Under our optimized conditions, a 23 base-pair single-stranded DNA sequence of the Verotoxin gene (VT2) of Escherichia coli, previously multiplied using PCR (40 cycles), could be detected within 10 minutes.
Tsuruoka
M
M
Advanced Science and Technology Laboratory, City of Hiroshima.
Honda
T
T
Karube
I
I
D001427:Bacterial Toxins / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis*; D019453:Escherichia coli O157 / Q000235:genetics*; D005454:Fluorescence Polarization; D009693:Nucleic Acid Hybridization; D022622:Shiga Toxin 1; D013997:Time Factors
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D022622:Shiga Toxin 1