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In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify <jats:italic>B<\/jats:italic>. <jats:italic>pseudomallei<\/jats:italic> DNA from clinical isolates. Through <jats:italic>in silico<\/jats:italic> search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to <jats:italic>B<\/jats:italic>. <jats:italic>pseudomallei<\/jats:italic>, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of <jats:italic>B<\/jats:italic>. <jats:italic>pseudomallei<\/jats:italic> 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 <jats:italic>B<\/jats:italic>. <jats:italic>pseudomallei<\/jats:italic> DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.<\/jats:p>","DOI":"10.1371\/journal.pntd.0010659","type":"journal-article","created":{"date-parts":[[2022,8,29]],"date-time":"2022-08-29T17:23:04Z","timestamp":1661793784000},"page":"e0010659","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":15,"title":["Highly specific and sensitive detection of Burkholderia pseudomallei genomic DNA by CRISPR-Cas12a"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3169-5509","authenticated-orcid":true,"given":"Somsakul 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