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

Abstract

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.

Benchmarking CRISPR-BP34 for point-of-care melioidosis detection in LMIC: a molecular diagnostics study

“…Study 2 was a diagnostic evaluation of the sensitivity and specificity of a CRISPR-BP34 prototype assay 20 conducted at Sunpasitthiprasong Hospital between May 2022 and December 2022 (Figure 1B). Minimal sample size was determined using the formula n = z 2 p(1-p) /d 2 where "z" is the 95% confidence interval at 1•96; "p" is the prevalence at 0•5; and "d" represents the margin of error at 0•1.…”

“…Bacterial genomic DNA was then extracted from the pellet using either hot alkaline lysis or a spin column, depending on the pellet size. B. pseudomallei DNA was amplified in an RPA reaction, and the resulting amplicons were added into a 50-μL CRISPR reaction, comprising of CRISPR RNA (crBP34) 20 , LbCas12a protein and FAM-biotin probes. This reaction was incubated at 37 °C for 60 minutes, after which a HybriDetect lateral flow dipstick (TwistDx, UK) was directly immersed into the reaction and allowed to develop for 5 minutes before reading by eye.…”

“…Not every sample cultured from a melioidosis patient yields positive result, a scenario driven by different local concentration of B. pseudomallei. Discordant results between culture and the CRISPR assay were tested by quantitative PCR (qPCR) using three primer sets listed in the appendix (pp 9, [19][20]. Given a large discrepancy in reported diagnostic sensitivity of PCR primers 18 and a high sequence diversity of B. pseudomallei, the use of primer combinations ensured an increased coverage of the detection.…”

“…This approach has been applied to other bacterial pathogens including Mycobacterium tuberculosis 19 and has been demonstrated to improve diagnosis and treatment responses. We previously described a robust CRISPR-based detection of genomic DNA of B. pseudomallei in vitro 20 . Here, we addressed the issues surrounding delayed diagnosis of melioidosis, established a diagnostic protocol for our recently developed CRISPR-Cas12a system (here-after termed CRISPR-BP34) 20 , and determined its diagnostic sensitivity and specificity.…”

“…We previously described a robust CRISPR-based detection of genomic DNA of B. pseudomallei in vitro 20 . Here, we addressed the issues surrounding delayed diagnosis of melioidosis, established a diagnostic protocol for our recently developed CRISPR-Cas12a system (here-after termed CRISPR-BP34) 20 , and determined its diagnostic sensitivity and specificity. Our results showed higher sensitivity and shortened diagnostic time of the CRISPR-BP34 detection compared with the gold standard culture.…”