Optimal probe length and target location for electrochemical detection of selected uropathogens at ambient temperature
Optimal probe length and target location for electrochemical detection of selected uropathogens at ambient temperature
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DOI：
10.1128/jcm.00423-08
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发表时间：
2008-08-01
影响因子：
9.4
通讯作者：
Haake, David A.
中科院分区：
文献类型：
--
作者：
Mastali, Mitra;Babbitt, Jane T.;Haake, David A.
我们先前已经证明了使用DNA生物传感器快速检测尿路病原体的临床有效性。该测定涉及捕获和检测探针对与细菌16 S rRNA靶分子的杂交，以在传感器表面上形成DNA-RNA夹心。辣根过氧化物酶偶联抗体与检测器探针结合，以酶促放大杂交信号。这些先前的研究涉及细菌16 S rRNA靶序列与35-mer寡核苷酸探针对在65 ℃下的杂交。环境温度检测将极大地促进即时技术的实现。本研究的目的是使用20至25摄氏度的杂交温度检查探针长度和靶位置对信号强度的影响。发现信号强度随16 S rRNA螺旋18的物种特异性凸起区域中的杂交位置而显著变化。长度短至10个核苷酸的探针对能够产生显著的电化学信号，并且对于长度为10至20个核苷酸的探针，信号强度与探针长度相关。大肠杆菌特异性15-mer探针对的灵敏度为约330个细胞。这些较短的探针允许区分肺炎克雷伯氏菌和奇异变形杆菌16 S rRNA靶序列，其差异仅为单个核苷酸。一组长度为11至23个核苷酸的寡核苷酸探针对能够区分七组尿路病原体。总之，我们已经开发了短的寡核苷酸探针对，用于在环境温度下使用电化学传感器进行尿路病原体的物种特异性鉴定。
We have previously demonstrated the clinical validity of the rapid detection of uropathogens by use of a DNA biosensor. This assay involves the hybridization of capture and detector probe pairs with bacterial 16S rRNA target molecules to form a DNA-RNA sandwich on the sensor surface. Horseradish peroxidase-conjugated antibody binds to the detector probe to enzymatically amplify the hybridization signal. These previous studies involved the hybridization of bacterial 16S rRNA target sequences with 35-mer oligonucleotide probe pairs at 65 degrees C. Achievement of point-of-care technology will be greatly facilitated by ambient-temperature detection. The purpose of this study was to examine the effects of probe length and target location on signal intensity using hybridization temperatures of 20 to 25 degrees C. Signal intensity was found to vary dramatically with hybridization location in the species-specific bulge region of 16S rRNA helix 18. Probe pairs of as short as 10 nucleotides in length were able to produce a significant electrochemical signal, and signal intensity was correlated with probe length for probes of 10 to 20 nucleotides in length. The sensitivity of the Escherichia coli-specific 15-mer probe pairs was approximately 330 cells. These shorter probes allowed differentiation of Klebsiella pneumoniae from Proteus mirabilis 16S rRNA target sequences differing by a single nucleotide. A panel of oligonucleotide probe pairs ranging from 11 to 23 nucleotides in length was able to distinguish among seven groups of urinary tract pathogens. In conclusion, we have developed short oligonucleotide probe pairs for the species-specific identification of uropathogens at ambient temperature by use of an electrochemical sensor.