Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance.
A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5'-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5'-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P<0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.
Wu
Vivian C H
VC
Department of Food Science and Human Nutrition, University of Maine, Orono, ME 04469-5735, USA.
Chen
Sz-Hau
SH
Lin
Chih-Sheng
CS
D018829:Adhesins, Bacterial / Q000235:genetics; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis*; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000235:genetics; D009841:Oligonucleotides / Q000032:analysis; D016133:Polymerase Chain Reaction; D011791:Quartz; D012680:Sensitivity and Specificity
D018829:Adhesins, Bacterial; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; C073819:eaeA protein, E coli; D011791:Quartz