Specific Multiplex Analysis of Pathogens Using a Direct 16S rRNA Hybridization in Microarray System

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• 作者：Byeong Hee Hwang ; Hwa Hui Shin ; Jeong Hyun Seo ; Hyung Joon Cha
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：June 5, 2012
• 年：2012
• 卷：84
• 期：11
• 页码：4873-4879
• 全文大小：409K
• 年卷期：v.84,no.11(June 5, 2012)
• ISSN：1520-6882

文摘

For the rapid multiplex analysis of pathogens, 16S rRNAs from cell lysates were directly applied onto a DNA microarray at room temperature (RT) for RNA鈥揇NA hybridization. To eliminate the labeling step, seven fluorescent-labeled detector probes were cohybridized with 16S rRNA targets and adjacent specific capture probes. We found that eight pathogens were successfully discriminated by the 16S rRNA-based direct method, which showed greater specificity than the polymerase chain reaction (PCR)-labeled method due to chaperone and distance effects. A new specificity criterion for a perfect match between RNA and DNA was suggested to be 21鈥?1% dissimilarity using correlation analysis between the mismatch and the sequence according to the guanine鈥揷ytosine (GC) percentage or the distribution of mismatches. Six categories of food matrix (egg, meat, milk, rice, vegetable, and mixed) were also tested, and the target pathogen was successfully discriminated within statistically significant levels. Finally, we found that the intrinsic abundance of 16S rRNA molecules successfully substituted PCR-based amplification with a low limit of detection of 10鈥?0³ cells mL^鈥? and a high quantitative linear correlation. Collectively, our suggested 16S rRNA-based direct method enables the highly sensitive, specific, and quantitative analysis of selected pathogens at RT within 2 h, even in food samples.