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. Author summary Melioidosis is a fatal infectious disease caused by a Gram-negative bacterium called Burkholderia pseudomallei. The bacteria can be found in many parts of the world, especially in the tropical and subtropical regions. Infection displays a variety of symptoms such as pneumonia, organ abscess and septicemia. The latter can lead to death within 24–48 hours if not properly diagnosed and treated. Rapid and accurate diagnosis, consequently, are essential for saving patients’ lives. Currently, culturing B. pseudomallei is a gold standard diagnostic method, but the assay turnaround time is 2–4 days, and the result could be of low sensitivity. Other detection methods such as real-time PCR and serological assays are limited by availability of equipment and by low specificity in endemic areas, respectively. For these reasons, in this study we developed a specific, sensitive and rapid detection assay for B. pseudomallei DNA, that is based on CRISPR-Cas12a system. The CRISPR-Cas12a is a protein-RNA complex that recognizes DNA. The RNA can be reprogramed to guide the detection of any DNA of interest, which in our case B. pseudomallei genomic DNA. Our data showed that this assay exhibited a 100% specificity to B. pseudomallei while discriminating against 10 other pathogens and human. The assay can detect B. pseudomallei DNA in less than one hour and does not require sophisticated equipment.