Establishment of a Real-Time Recombinase Polymerase Amplification for Rapid Detection of Pathogenic Yersinia enterocolitica

Abstract

Yersinia enterocolitica is a zoonotic proto-microbe that is widespread throughout the world, causes self-limiting diseases in humans or animals and even leads to sepsis and death in patients with severe cases. In this study, a real-time recombinase polymerase amplification (RPA) assay for pathogenic Y. enterocolitica was established based on the ail gene. The results showed that the RPA detection for Y. enterocolitica could be completed within 20 min at an isothermal temperature of 38 °C by optimizing the conditions in the primers and Exo probe. Moreover, the sensitivity of the current RT-RPA was 10^-4 ng/μL, and the study found that the assay was negative in the application of the genomic DNA of other pathogens. These suggest the establishment of a rapid and sensitive real-time RPA method for the detection of pathogenic Y. enterocolitica, which can provide new understandings for the early diagnosis of the pathogens.

Keywords: Yersinia enterocolitica; ail gene; rapid detection; recombinase polymerase amplification.

Figure 1

Screening of optimal real-time RPA primer–probe combinations. (a) Screening the optimal primers for the real-time RPA by the basic RPA: 1, NTC; 2, F1/R1; 3. F2/R2; 4, F3/R3; 5, F1/R2; (b) Screening the optimal primers for the real-time RPA: 1, F3/R3; 2, F1/R1; 3, F2/R2; 4, F1/R2; 5, F1/R3; 6, F2/R3; 7, NTC; (c) Screening the optimal probe for the real-time RPA: 1, Probe-5; 2, Probe-6; 3. NCT.

Figure 2

Sensitivity and specificity of real-time RPA assay. (a) Sensitivity test of real-time RPA assay: 1, 10 ng/μL; 2, 10⁰ ng/μL; 3, 10⁻¹ ng/μL; 4, 10⁻² ng/μL; 5, 10⁻³ ng/μL; 6, 10⁻⁴ ng/μL; 7, 10⁻⁵ ng/μL; 8, NTC. (b) Specificity test of real-time RPA assay: 1, Y. enterocolitica ATCC 23715; 2, Y. enterocolitica ZDN6; 3, Pasteurella P0810; 4, Shigella Castellani 06-01-03; 5, Slmonella 06-03-01; 6, Escherichia coli 03-03; 7, Aeromonas hydrophila AH 1302; 8, Bacillus cereus 06-05-02; 9, non-pathogenic Y. enterocolitica GN 22; 10, Staphylococcus aureus 04-011; 11, NTC.

Figure 3

Repeatability of the real-time RPA assay. (a–c) Repeatability of the real-time RPA assay with different groups: 1–3, 10⁰ ng/μL; 4–6, 10⁻² ng/μL; 7–9, 10⁻⁵ ng/μL; 10–12, NTC.

Figure 4

Validation of the real-time RPA assay: 1, pathogenic Y. enterocolitica ZDN 6 (Tt 3.17); 2, pathogenic Y. enterocolitica ATCC 23715 (Tt 9.51); 3, pathogenic Y. enterocolitica 08-01 (Tt 10.02); 4, pathogenic Y. enterocolitica GN 22 (Tt 18.55); 5, pathogenic Y. enterocolitica ZDN 22 (Tt 22.47); 6, non-pathogenic Y. enterocolitica GLN 1 (Tt undetermined); 7, non-pathogenic Y. enterocolitica HNN 18 (Tt undetermined); 8, non-pathogenic Y. enterocolitica HNN 47 (Tt undetermined); 9, non-pathogenic Y. enterocolitica ZDN 37 (Tt undetermined); 10, non-pathogenic Y. enterocolitica HNN 63 (Tt undetermined); 11, NTC (Tt undetermined).