College of Environmental Science and Engineering, Key Laboratory of Environmental Biology and Pollution Control, Ministry of Education, Hunan University, Changsha 410082, China.
Anal Chim Acta. 2010 Apr 1;664(1):95-9. doi: 10.1016/j.aca.2010.01.064. Epub 2010 Feb 6.
A two-probe tandem DNA hybridization assay based on time-resolved fluorescence was employed to detect Escherichia coli strain. The amino modified capture probe was covalently immobilized on the common glass slide surface. The Eu(TTA)(3)(5-NH(2)-phen) with the characteristics of long lifetime and intense luminescence was labeled with reporter probe. The original extracted DNA samples without the purification and amplification process were directly used in the hybridization assay. The concentration of capture probe, hybridization temperature, hybridization and washing time were optimized. The detection limit is about 1.49x10(3) CFU mL(-1) E. coli cells, which is comparable to the value of most microbiology methods. The proposed method has the advantages of easy operation, satisfactory sensitivity and specificity, which can provide a promising technique for monitoring the microorganisms.
基于时间分辨荧光的双探针串联 DNA 杂交分析方法被用于检测大肠杆菌菌株。经氨基修饰的捕获探针被共价固定在普通玻片表面。具有长荧光寿命和强发光特性的 Eu(TTA)(3)(5-NH(2)-phen)被标记为报告探针。原始提取的 DNA 样本无需纯化和扩增过程，即可直接用于杂交分析。优化了捕获探针浓度、杂交温度、杂交和洗涤时间。检测限约为 1.49x10(3) CFU mL(-1)大肠杆菌细胞，与大多数微生物学方法的检测限相当。该方法具有操作简单、灵敏度和特异性好等优点，可为监测微生物提供一种有前途的技术。
