Detection of with CRISPR-Cas12a based on specific sequence tags.

Melioidosis is a bacterial infection caused by (), posing a significant threat to public health. Rapid and accurate detection of is crucial for preventing and controlling melioidosis. However, identifying is challenging due to its high similarity to other species in the same genus. To address this issue, this study proposed a dual-target method that can specifically identify in less than 40 min. We analyzed 1722 genomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguish from its closely related species. Specifically, we selected two specific tags, LC1 and LC2, which we combined with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas12a) system and recombinase polymerase amplification (RPA) pre-amplification. Our analysis showed that the dual-target RPA-CRISPR/Cas12a assay has a sensitivity of approximately 0.2 copies/reaction and 10 fg genomic DNA for LC1, and 2 copies/reaction and 20 fg genomic DNA for LC2. Additionally, our method can accurately and rapidly detect in human blood and moist soil samples using the specific sequence tags mentioned above. In conclusion, the dual-target RPA-CRISPR/Cas12a method is a valuable tool for the rapid and accurate identification of in clinical and environmental samples, aiding in the prevention and control of melioidosis.