A rapid and ultrasensitive dual detection platform based on Cas12a for simultaneous detection of virulence and resistance genes of drug-resistant Salmonella.

Accurate, sensitive, and rapid detection of Salmonella and determination of whether it carries drug resistance genes plays an important role in guiding the clinical medication of salmonellosis and laying a foundation for studying the mechanism of drug resistance transmission of Salmonella. Here, a novel nontransferable, ultrasensitive dual detection platform (Cas12a-Ddp) was developed. The round cap allowed for temporary storage of more Cas12a detection solution than flat cap, enabling one-pot assays and reducing aerosol contamination. The results were read out in dual mode by the microplate reader and UV visualization to achieve sensitive dual-target detection of the virulence genes and drug resistance genes of Salmonella simultaneously, with the possibility of onsite detection. Cas12a-Ddp was combined with multiple polymerase chain reactions and recombinase polymerase amplifications successively. An ultrasensitive dual detection limit of 1 CFU/mL was obtained without any cross-reaction within 40 min. This was an improvement of 1-2 orders of magnitude over the existing methods. Cas12a-Ddp overcame the influence of proteins and fat in liquid matrix foods. It was used for the detection of drug-resistant Salmonella in milk and skim milk powder, also with the dual detection limit of 1 CFU/mL and spiked recovery of 68.58%-158.49%. It was also used for the analysis of Salmonella resistance rate analysis. The Cas12a-Ddp provided a reliable, fast, sensitive, and practical multi-CRISPR detection platform.