Construction of an RPA-CRISPR visualization system for rapid detection of Burkholderia mallei.

BACKGROUND: Glanders is a chronic zoonosis caused by Burkholderia mallei (BM). The main sources of infection are infected horses, mules, donkeys and other single-hoofed equine animals. There is currently no commercial vaccine for glanders, so the mortality rate of animals with this disease is extremely high. The prevalence of the disease is due mainly to improper border trade quarantine, so early diagnosis of the disease is crucial for epidemic prevention and control. RESULTS: In this study, a one-step isothermal amplification detection method based on RPA and CRISPR was developed for BM targets. Through sgRNA design and verification and RPA primer design and screening, BM-sgRNA-3 and BM-RPA-F2/R1 were selected for the construction of the test system. The constructed RPA-CRISPR detection system has a detection limit of 1E1 copies/T and good specificity. On this basis, one-step visualization and test strip detection methods were further established. The results showed that the RPA-CRISPR one-step visualization process can achieve stable detection at a concentration of 1E1 copies/T, and the sensitivity of the RPA-CRISPR test strip detection system can reach 1E2 copies/T. CONCLUSIONS: This study established a one-step RPA–CRISPR assay specifically targeting Burkholderia mallei and translated it into both fluorescence-based and lateral flow formats. The assay demonstrated high analytical sensitivity and specificity, and its simple workflow and visual readout make it suitable for routine diagnosis and on-site screening of glanders.