High-throughput microfluidic strategy based on RAA-CRISPR/Cas13a dual signal amplification for accurate identification of pathogenic Listeria

Listeria spp. is a kind of important food-borne pathogen with a high fatality rate and is widely distributed in food chain, posing a potential threat to the public health. It is urgently necessary to develop a pathogenic Listeria detection platform with high specificity, sensitivity, high-throughput, and user-friendliness to meet the re-quirements of on-line monitoring of food production. We therefore developed a one-step detection method (hMC-CRISPR) based on recombinase aided amplification (RAA) and CRISPR on a high-throughput microfluidic chip. This strategy coordinates the nucleic acid amplification and the cleavage efficiency of CRISPR/Cas13a through the transcription process, thereby preventing the complete cleavage of all genomic DNA and amplicons from being cleaved. The corresponding RAA-CRISPER/Cas13a system was designed based on the specific markers of Listeria spp., L. monocytogenes, L. innocua and L. ivanovii, and the reaction volume was limited to 5 mu L to match the microfluidic chip. This strategy achieved sensitive and robust detection of pathogenic Listeria with a sample-to-answer time of ~60 min, and a detection limit of each index reached aM level. More importantly, hMC-CRISPR can detect pathogenic Listeria in eight samples at one time and avoid the influence of aerosol on the test results. It is practically validated by quantitatively determining four types of spiked samples and 24 natural samples. Collectively, this new strategy has great potential to improve the current rapid screening of pathogenic Listeria.