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

• Published in: Biosensors & bioelectronics Vol. 52; pp. 433 - 437
• Main Authors: Duy, Janice, Smith, Rosemary L, Collins, Scott D, Connell, Laurie B
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 15.02.2014; Elsevier
• Subjects: Biological and medical sciences; Biological Assay; Biosensing Techniques - methods; Biosensor; Biosensors; Biotechnology; Carbocyanines - chemistry; Colorimetric; Colorimetry; Field-compatible; Fundamental and applied biological sciences. Psychology; Harmful Algal Bloom; Hybridization; Methods. Procedures. Technologies; Nucleic Acid Hybridization; Peptide nucleic acid; Peptide Nucleic Acids - chemistry; RNA, Ribosomal - chemistry; RNA, Ribosomal - isolation & purification; rRNA; Various methods and equipments; Field-compatible; Peptide nucleic acid; Hybridization; rRNA; Biosensor; Colorimetric; Rapid technique; Bioassay; Ribosomal RNA; Colorimetry; Detection
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2012.05.039

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. ► Rapid RNA hybridization is achieved with PNA probes and a symmetrical cyanine dye. ► Single base-mismatched RNA are digested off the probe with micrococcal nuclease. ► Unamplified target RNA is quantified in mixtures of related non-target sequences. ► A handheld colorimeter was designed to make the assay field-compatible. ► The colorimeter performance is comparable to a laboratory spectrophotometer.