Wu Vivian C H, Chen Sz-Hau, Lin Chih-Sheng
Department of Food Science and Human Nutrition, University of Maine, Orono, ME 04469-5735, USA.
Biosens Bioelectron. 2007 Jun 15;22(12):2967-75. doi: 10.1016/j.bios.2006.12.016. Epub 2007 Jan 16.
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.
本研究开发了一种用于在循环流动系统中实时检测大肠杆菌O157:H7的DNA压电生物传感方法。使用用于检测大肠杆菌O157:H7基因eaeA的特异性探针[一种带有或不带有额外12个脱氧胸苷5'-单磷酸（12-dT）的30聚体寡核苷酸]、合成寡核苷酸靶标（30和104聚体）以及来自大肠杆菌O157:H7 eaeA基因的PCR扩增DNA片段（104 bp），评估探针在循环流动石英晶体微天平（QCM）装置中固定和与靶DNA杂交的效率。发现探针5'-末端的硫醇修饰对于探针固定在QCM装置的金表面至关重要。向探针中添加12-dT作为间隔物，显著提高了（P<0.05）杂交效率（H%）。结果表明，当探针与30聚体和104聚体靶标杂交时，间隔物分别将H%提高了1.4倍和2倍。间隔物减少了载体对固定化寡核苷酸杂交行为的空间干扰，特别是当探针与相对较长的寡核苷酸靶标杂交时。QCM系统也应用于检测来自大肠杆菌O157:H7实际样品的PCR扩增DNA。所得PCR扩增双链DNA的H%与合成靶标T-104AS（一种单链DNA）相当。该压电生物传感系统具有进一步应用的潜力。这种方法为将该方法纳入基于PCR的快速DNA分析集成系统奠定了基础。
