Specific Multiplex Analysis\nof Pathogens Using a Direct\n16S rRNA Hybridization in Microarray System

Abstract

For the rapid multiplex analysis of pathogens, 16S rRNAs\nfrom cell\nlysates were directly applied onto a DNA microarray at room temperature\n(RT) for RNA–DNA hybridization. To eliminate the labeling step,\nseven fluorescent-labeled detector probes were cohybridized with 16S\nrRNA targets and adjacent specific capture probes. We found that eight\npathogens were successfully discriminated by the 16S rRNA-based direct\nmethod, which showed greater specificity than the polymerase chain\nreaction (PCR)-labeled method due to chaperone and distance effects.\nA new specificity criterion for a perfect match between RNA and DNA\nwas suggested to be 21–41% dissimilarity using correlation\nanalysis between the mismatch and the sequence according to the guanine–cytosine\n(GC) percentage or the distribution of mismatches. Six categories\nof food matrix (egg, meat, milk, rice, vegetable, and mixed) were\nalso tested, and the target pathogen was successfully discriminated\nwithin statistically significant levels. Finally, we found that the\nintrinsic abundance of 16S rRNA molecules successfully substituted\nPCR-based amplification with a low limit of detection of 10–10³ cells mL^–1 and a high quantitative linear\ncorrelation. Collectively, our suggested 16S rRNA-based direct method\nenables the highly sensitive, specific, and quantitative analysis\nof selected pathogens at RT within 2 h, even in food samples.