A field-deployable colorimetric bioassay for the rapid and specific detection of ribosomal RNA

Abstract

Rapid and specific on-site detection of disease-causing or toxin-producing organisms is essential to public health and safety. Many molecular recognition methods target ribosomal RNA sequences due to their specificity and abundance in the cell. In this work RNA targets were identified and quantified using a colorimetric bioassay. Peptide nucleic acid (PNA) probes were used to capture RNA targets, and a micrococcal nuclease digestion was performed to remove all non-target nucleic acids, including single base mismatches flanked by adenines or uracils. Perfectly-matched PNA-RNA hybrids remained intact and were detected using the symmetrical cyanine dye 3,3'-diethylthiadicarbocyanine iodide (DiSC2(5)). Assay applicability to complex samples was demonstrated using mixtures containing RNA sequences from two related, harmful algal bloom-causing Alexandrium species. Target RNA was detected even in mixtures with mismatched sequences in excess of the perfect match. The fieldability of the assay was tested with a portable two-wavelength colorimeter developed to quantify the dye-indicated hybridization signal. The colorimeter sensing performance was shown to be comparable to a laboratory spectrophotometer. This quick, inexpensive and robust system has the potential to replace laborious identification schemes in field environments.

Keywords: Biosensor; Colorimetric; Field-compatible; Hybridization; Peptide nucleic acid; rRNA.