关键词: 布鲁氏菌, RAA, DETECTOR系统, SHERLOCK系统, AsCas12a, LwaLwaCas13a

Abstract:

The purpose of this study is to establish efficient and rapid detection systems for brucellosis that does not rely on professional equipment and personnel, and finished rapid detection of clinical samples within 40 minutes. First, the genomes of different Brucellaspecies were compared, and the core consensus sequence of Brucella was found. The positive plasmid was constructed using the BrucellaS2 strain gene SEQ NO.6 (CN 105018489A) as a template for subsequent experiments; CRISPR/AsCas12a and LwaCas13a proteins were expressed by prokaryotic expression methods; the recombinase-mediated isothermal nucleic acid amplification technology (RAA) and in vitro expression technology combined with the AsCas12a/LwaCas13a DETECTOR system were used to optimize the efficient detection system and the detection was determined by the infinite dilution method. The designed RAA primers can effectively amplify DNA fragments in samples. Optimal DETECTOR systems obtained 200 nmol·L^-1 AsCas12a protein, 200 nmol·L^-1 gRNA; also needs 300 nmol·L^-1 LwaCas13a and 400nM crRNA. The optimal reaction time of the detection system of RAA-AsCas12a and RAA-LwaCas13a was 35 min, the optimal detection time of the lateral flow strip was 20 min, the minimum detection concentration of bacteria was 10 Copies ·μL^-1, and it had good detection specificity. The detection test of clinical samples showed both methods had good repeatability and detection accuracy, but LwaCas13a had higher precision, but there was no significant difference between the two methods. This study established two RAA-CRISPR/Cas Brucellanucleic acid rapid detection systems, which can be well applied in clinical, and the RAA-LwaCas13a detection method has higher accuracy.

Key words: Brucella, RAA, DETECTOR system, SHERLOCK system, AsCas12a, LwaLwaCas13a