Simple and rapid bioluminescent detection of two verotoxin genes using allele-specific PCR of E. coli O157: H7.
Allele-specific PCR for E. coli O157 was conducted with primers specific to verotoxin genes, verotoxin 1 (VT1) and verotoxin 2 (VT2). VT is an important cause of haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) worldwide. We developed a simple, rapid bioluminescent detection method for E. coli O157. The method is based on the determination of pyrophosphoric acid (PPi) released during allele-specific PCR. Thus, released PPi is converted to ATP by ATP sulphurylase and the concentration of ATP is determined using the firefly luciferase reaction. As a result, VT1, VT2 and DNA with VT1/VT2 were clearly identified by this method. This protocol, which does not require expensive equipment, can be utilized to monitor the PCR product rapidly. Additionally, this methodology can be used as a high-throughput approach for measuring PCR products.
Imamura
O
O
School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo 142-8555, Japan.
Arakawa
H
H
Maeda
M
M
D000483:Alleles; D002138:Calibration; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D011756:Diphosphates / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D019453:Escherichia coli O157 / Q000235:genetics*; D049454:Firefly Luciferin / Q000737:chemistry; D007202:Indicators and Reagents; D008156:Luciferases / Q000737:chemistry; D049449:Luminescence; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022603:Shiga Toxins / Q000235:genetics*; D013430:Sulfate Adenylyltransferase / Q000737:chemistry
D017931:DNA Primers; D004269:DNA, Bacterial; D011756:Diphosphates; D007202:Indicators and Reagents; D022603:Shiga Toxins; C107241:diphosphoric acid; D049454:Firefly Luciferin; D008156:Luciferases; D013430:Sulfate Adenylyltransferase