Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a

Detection of Burkholderia pseudomallei , a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B . pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B . pseudomallei , named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B . pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B . pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.