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

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 degrees 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/mu L and 10(1) 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 10(1) CFU/mL, 10(2) CFU/mL, and 10(2) 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.

Automated summary

In this study, a rapid and sensitive method for detecting Burkholderia gladioli in food was developed using the CRISPR/Cas12a system combined with recombinant enzyme polymerase amplification (RPA). The optimized RPA-CRISPR/Cas12a assay was able to specifically detect Burkholderia gladioli at a constant temperature without the need for large equipment. The method showed a detection limit of 10(-3) ng/μL at the genomic DNA level and 10(1) CFU/mL at the bacterial quantity level. Real food samples, including rice noodles, fresh white noodles, and glutinous rice flour, were tested, and the method showed a detection limit of 10(1) CFU/mL, 10(2) CFU/mL, and 10(2) CFU/mL, respectively, without any enrichment steps. The entire detection process, including sample pretreatment and DNA extraction, could be completed within one hour. Compared to the qPCR method, the RPA-CRISPR/Cas12a method was simpler and more sensitive, allowing for rapid and easy visual detection of Burkholderia gladioli suitable for field detection.