Rapid and Simple Detection of Burkholderia gladioli in Food Matrices Using RPA-CRISPR/Cas12a Method

Abstract

Pathogenic variants of Burkholderia gladioli pose a serious threat to human health and food safety, but there is a lack of rapid and sensitive field detection methods for Burkholderia gladioli. In this study, the CRISPR/Cas12a system combined with recombinant enzyme polymerase amplification (RPA) was used to detect Burkholderia gladioli in food. The optimized RPA-CRISPR/Cas12a assay was able to specifically and stably detect Burkholderia gladioli at a constant 37 °C without the assistance of large equipment. The detection limit of the method was evaluated at two aspects, the genomic DNA (gDNA) level and bacterial quantity, of which there were 10−3 ng/μL and 101 CFU/mL, respectively. Three kinds of real food samples were tested. The detection limit for rice noodles, fresh white noodles, and glutinous rice flour samples was 101 CFU/mL, 102 CFU/mL, and 102 CFU/mL, respectively, without any enrichment steps. The whole detection process, including sample pretreatment and DNA extraction, did not exceed one hour. Compared with the qPCR method, the established RPA-CRISPR /Cas12a method was simpler and even more sensitive. Using this method, a visual detection of Burkholderia gladioli that is suitable for field detection can be achieved quickly and easily.