Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA

Quenched autoligation (QUAL) probes are a class of self-reacting nucleic acid probes that give strong fluorescence signal in the presence of fully complementary RNAs and selectivity against single nucleotide differences in solution. Here, we describe experiments designed to test whether QUAL probes can discriminate between bacterial species by the detection of small differences in their 16S rRNA sequences. Probes were introduced into live cells using small amounts of detergent, thus eliminating the need for fixation, and fluorescence signal was monitored both by microscopy and by flow cytometry without any washing steps. The effects of probe length, modified backbone, probe concentration and growth state of the bacteria were investigated. The data demonstrate specific fluorescence discrimination between three closely related bacteria, Escherichia coli, Salmonella enterica and Pseudomonas putida, based on single nucleotide differences in their 16S rRNA. Discrimination was possible with cells in mid-log phase or in lag phase. These results suggest that QUAL probes may be useful for rapid identification of microorganisms in laboratory and clinical settings.