Visual and sensitive detection of viable pathogenic bacteria by sensing of RNA markers in gold nanoparticles based paper platform

Abstract

Food-borne pathogens have been recognized as a major cause of human infections worldwide. Their identification needs to be simpler, cheaper and more reliable than the traditional methods. Here, we constructed a low-cost paper platform for viable pathogenic bacteria detection with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper-based platform to perform a visual test using sandwich hybridization assays. When the RNA products migrated along the platform by capillary action, the gold nanoparticles accumulated at the designated area. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from L. monocytogenes could be detected. It could also be used to specifically detect 20 CFU/mL L. monocytogenes from actual samples. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. This method is suitable for point-of-care applications to detect food-borne pathogens, as it can overcome the false-positive results caused by amplifying nonviable L. monocytogenes. Furthermore, the results can be imaged and transformed into a two-dimensional bar code through an Android-based smart phone for further analysis or in-field food safety tracking.

Keywords: Bioactive paper-based platform; Listeria monocytogenes; Pathogenic bacteria; RNA amplification.