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首页> 外文期刊>Journal of Clinical Microbiology >Optimal Probe Length and Target Location for Electrochemical Detection of Selected Uropathogens at Ambient Temperature
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Optimal Probe Length and Target Location for Electrochemical Detection of Selected Uropathogens at Ambient Temperature

机译：在环境温度下电化学检测选定的致病菌的最佳探针长度和靶标位置

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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°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°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.
机译：我们以前已经证明了通过使用DNA生物传感器快速检测尿毒症的临床有效性。该测定法涉及捕获和检测器探针对与细菌16S rRNA靶分子的杂交，以在传感器表面形成DNA-RNA三明治。辣根过氧化物酶偶联的抗体与检测器探针结合，以酶促方式放大杂交信号。这些先前的研究涉及在65°C下将细菌16S rRNA靶序列与35-mer寡核苷酸探针对杂交。环境温度检测将大大促进即时医疗技术的实现。这项研究的目的是使用20至25°C的杂交温度检查探针长度和目标位置对信号强度的影响。发现信号强度随杂交位置在16S rRNA螺旋18的物种特异性凸起区域中发生显着变化。短至10个核苷酸的探针对能够产生显着的电化学信号，并且信号强度与探针长度相关用于长度为10到20个核苷酸的探针。大肠埃希氏菌特异性15聚体探针对的敏感性约为330个细胞。这些较短的探针可以使肺炎克雷伯菌和特异变形杆菌16S rRNA靶序列的区别在于单个核苷酸。一组长度在11至23个核苷酸之间的寡核苷酸探针对能够区分7组泌尿道病原体。总之，我们开发了短的寡核苷酸探针对，用于通过使用电化学传感器在环境温度下对尿毒症菌进行物种特异性鉴定。

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