Establishment of a Simple, Sensitive, and Specific Salmonella Detection Method Based on Recombinase-Aided Amplification Combined with dsDNA-Specific Nucleases

Abstract

Salmonella is a common foodborne pathogen that can cause food poisoning, posing a serious threat to human health. Therefore, quickly, sensitively, and accurately detecting Salmonella is crucial to ensuring food safety. For the Salmonella hilA gene, we designed Recombinase-aided amplification (RAA) primers and dsDNA-specific nuclease (DNase) probes. The ideal primer and probe combination was found when conditions were optimized. Under UV light, a visual Salmonella detection technique (RAA-dsDNase) was developed. Additionally, the RAA-dsDNase was modified to further reduce pollution hazards and simplify operations. One-pot RAA-dsDNase-UV or one-pot RAA-dsDNase-LFD was developed as a Salmonella detection method, using UV or a lateral flow dipstick (LFD) for result observation. Among them, one-pot RAA-dsDNase and one-pot RAA-dsDNase-LFD had detection times of 50 min and 60 min, respectively, for detecting Salmonella genomic DNA. One-pot RAA-dsDNase-UV had a detection limit of 10¹ copies/μL and 10¹ CFU/mL, while one-pot RAA-dsDNase-LFD had a sensitivity of 10² copies/μL and 10² CFU/mL. One-pot RAA-dsDNase-UV and one-pot RAA-dsDNase-LFD assays may identify 17 specific Salmonella serovars witho ut causing a cross-reaction with the remaining 8 bacteria, which include E. coli. Furthermore, Salmonella in tissue and milk samples has been reliably detected using both approaches. Overall, the detection method developed in this study can quickly, sensitively, and accurately detect Salmonella, and it is expected to become an important detection tool for the prevention and control of Salmonella in the future.

Keywords: RAA; Salmonella; dsDNase; one pot; visualization.

Figure 1

Schematic diagram and feasibility analysis of RAA-dsDNase detection for Salmonella. (A) The principle of RAA-dsDNase detection for Salmonella mainly includes three parts. (I) dsDNase has no degrading activity on ssDNA fluorescent probe. (II) When dsDNase was absent from the reaction system, the ssDNA fluorescent probe remains intact. (III) When the template DNA was amplified by RAA, then dsDNase was added to the system, and the ssDNA fluorescent probe was degraded and released a fluorescent signal. (B,C) Feasibility analysis of RAA-dsDNase detection for Salmonella.

Figure 2

Sensitivity and specificity analysis of RAA-dsDNase. (A,B) The detection limit of RAA-dsDNase was 101 copies/μL (plasmid) and 101 CFU/mL (bacteria). (C) When the genomic DNA of Salmonella exists, RAA-dsDNase releases a strong fluorescent signal, while in the presence of genomic DNA of other species, including Escherichia coli, no fluorescent signal was produced. Strain numbers correspond to the details in Table S1.

Figure 3

Schematic diagram and feasibility analysis of one-pot RAA-dsDNase-UV and one-pot RAA-dsDNase-LFD detection for Salmonella. (A) Simultaneously adding the components of the RAA and dsDNase reactions into an EP tube, with RAA at the bottom of the tube and dsDNase and the fluorescent probe at the tube cap. After the RAA reaction was completed, it was briefly centrifuged, inverted to mix, and finally the results were observed using UV and LFD. (B,C) The feasibility analysis results showed that fluorescence or detection bands appeared only when the genomic DNA of Salmonella existed.

Figure 4

Sensitivity analysis of one-pot RAA-dsDNase-UV and one-pot RAA-dsDNase-LFD. (A,C) The detection limit of one-pot RAA-dsDNase-UV was 101 copies/μL (plasmid) and 101 CFUs (bacteria). (B,D) The detection limit of one-pot RAA-dsDNase-LFD was 102 copies/μL (plasmid) and 102 CFU/mL (bacteria).

Figure 5

Specificity analysis of one-pot RAA-dsDNase-UV and one-pot RAA-dsDNase-LFD. Specificity analysis results showed that one-pot RAA-dsDNase-UV and one-pot RAA-dsDNase-LFD could specifically detect Salmonella, without cross-reactivity to other strains of bacteria. Strain numbers correspond to the details in Table S1.

Figure 6

Detection of Salmonella in real samples. (A) Observation of liver tissue samples from sick and healthy chickens in clinical practice with the naked eye. The red arrow indicates the location of the nodule. (B) Traditional culture method was used to analyze Salmonella in the tissues of sick chickens, and the results showed typical morphology of Salmonella pullorum. (C) One-pot RAA-dsDNase-UV, one-pot RAA-dsDNase-LFD, and PCR analysis showed that significant fluorescent signals or detection bands were produced in the tissues of the hearts, livers, spleens, lungs, kidneys, and intestines of sick chicks, while no corresponding phenomenon was observed in healthy chicken tissues. (D) And similar results were obtained from PCR analysis. (E) Detection of Salmonella in milk using one-pot RAA-dsDNase-UV and one-pot RAA-dsDNase-LFD showed that both methods produced clear fluorescent signals or detection bands.