RPA-SYBR Green I based instrument-free visual detection for pathogenic Yersinia enterocolitica in meat

Abstract

Pathogenic Yersinia (Y.) enterocolitica is the primary causative agent of Yersiniosis, with outbreaks in numerous countries around the world, and causes diarrhea and vomiting in animals and humans. Therefore, an instrument-free and convenient nucleic acid visualization method, RPA-SYBR Green I, was established, which combines recombinase polymerase amplification (RPA) with the fluorescent dye SYBR Green I for the detection of the adhesion gene ail in pathogenic Y. enterocolitica. After optimization of a series of conditions such as primer concentration, the detection of pathogenic Y. enterocolitica could be finally completed within about 20 min (from DNA extraction to observation of results) at an isothermal temperature of 39°C. RPA-SYBR Green I had no cross-reactivity with other bacteria and the detection limit was 10¹ CFU/μL, with sensitivity equal to that of conventional PCR. The method established in this paper and conventional PCR identified a total of 5 spiked samples and 15 meat samples stored in refrigerated, and it was concluded that there was 100% consistency between the two methods. Overall, RPA-SYBR Green I is a visual and facilitate detection assay that can accurately discover pathogenic Y. enterocolitica.

Keywords: Instrument-free and visual detection; Pathogenic Yersinia enterocolitica; Recombinase polymerase amplification; SYBR Green I.