Recombinase Polymerase Amplification Coupled with a Photosensitization Colorimetric Assay for Fast <i>Salmonella</i> spp. Testing

Abstract

<i>Salmonella</i> spp. is one of the most serious foodborne pathogens causing millions of infection cases annually, especially in resource-limited areas. The standard culture method (2-3 days) and current nucleic acid amplification-based testing are not suitable for on-site testing in rural areas with heavy <i>Salmonella</i> spp. burden. Here, we developed a colorimetric recombinase polymerase amplification (RPA) method for fast and sensitive <i>Salmonella</i> spp. testing in 1 h. Specifically, the invA gene from the genomic DNA of <i>Salmonella</i> spp. was amplified isothermally to produce double-stranded DNA (dsDNA) amplicons, which were directly quantified by a photosensitization colorimetric assay. The proposed method offered the lowest detectable concentration of 5 × 10³ colony-forming units/mL (cfu/mL), which is much lower than that of ELISA (10⁵-10⁷ cfu/mL). The detectable limit could be further pushed down to 3 cfu/mL upon coupling with bacteria pre-enrichment for 6 h. Analysis of synthetic milk samples confirmed the high precision (90%) and specificity (95%) of the method for <i>Salmonella</i> spp. testing. Moreover, use of a DNA releaser could further simplify the whole testing operation. Because RPA features low-temperature amplification (25-42 °C) without the need for specific instruments and the dsDNA-based photosensitization colorimetric assay served as a simple and facile readout for RPA, our method thus allows fast and low-cost <i>Salmonella</i> spp. testing in food samples.