Recombinase Polymerase Amplification Coupled with a Photosensitization Colorimetric Assay for Fast <i>Salmonella</i> spp. Testing
Xianming Li, Ting Zheng, Yani Xie, Feng Li, Xia Jiang, Xiandeng Hou, Peng Wu
Abstract
Salmonella 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 Salmonella spp. burden. Here, we developed a colorimetric recombinase polymerase amplification (RPA) method for fast and sensitive Salmonella spp. testing in 1 h. Specifically, the invA gene from the genomic DNA of Salmonella 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 Salmonella 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 Salmonella spp. testing in food samples.