基于RAA-CRISPR/Cas12a建立牛病毒性腹泻病毒的可视化快速检测方法及其初步应用OA北大核心

To establish a visual detection method for bovine viral diarrhea virus(BVDV)based on the combination of recombinant enzyme mediated amplification(RAA)and CRISPR/Cas12a.By referring to the BVDV 5'UTR gene in the GenBank database,specific primers and probes for the RAA reaction were de-signed to further optimize the reaction conditions of RAA-CRISPR/Cas12a,and the detection method of RAA-CRISPR/Cas12a was evaluated by measuring sensitivity,specificity,and repeatability.BVDV was de-tected in 251 calf diarrhea samples by PCR and RAA-CRISPR/Cas12a,respectively.The results indicated that for the RAA-CRISPR/Cas12a detection method,the minimum detection limit of BVDV was 3 copies/μL and the detection time was 30 min.After multiple batches of positive samples with different copy num-bers were tested,the coefficients of variation intra-and inter-batches were both less than 5％and and the results of this method were negative in the detection of other common pathogens of cattle with good specificity.In clinical samples,the PCR positive rate of BVDV was 6.37％.When RAA-CRISPR/Cas12a was utilized simultaneously,the positive detection rate was 29.48％,and the positive detection rate reached 4.63 times.In this study,a rapid visual detection method for BVDV based on RAA-CRISPR/Cas12a was established,which is of great significance for the prevention and control of diarrhea in calves.