RAA-DETECTOR布鲁氏菌核酸快速检测方法的建立及应用

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关键词：布鲁氏菌;RAA;DETECTOR系统;SHERLOCK系统，AsCasl2a;LwaLwaCas13a中图分类号：S852.614 文献标志码：A 文章编号：0366-6964（2025）10-5115-10

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 4O minutes. First，the genomes of different Brucella species 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/AsCasl2a and LwaCasl3a proteins were expressed by prokaryotic expression methods； the recombinase-mediated isothermal nucleic acid amplification technology （RAA） and in vitro expression technology combined with the AsCasl2a/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 200nmol⋅L-1 AsCasl2a protein， 200nmol⋅L-1 gRNA;also needs 300nmol⋅L-1 LwaCas13a and 400nM crRNA. The optimal reaction time of the detection system of RAA-AsCasl2a and RAA-LwaCasl3a was 35min ，the optimal detection time of the lateral flow strip was 20min ，theminimum detection concentration of bacteria was 10Copies∙μL-1 ，and it had good detection specificity. The detection test of clinical samples showed both methods had good repeatability and detection accuracy， but LwaCasl3a had higher precision， but there was no significant difference between the two methods. This study established two RAA-CRISPR/Cas Brucella nucleic acid rapid detection systems，which can be well applied in clinical， and the RAA-LwaCasl3a detection method has higher accuracy.

Keywords： Brucella ；RAA；DETECTOR system；SHERLOCK system； AsCasl2a; LwaLwa-Casl3a

∗ Corresponding author： SU Feng，E-mail ： suf sdau.edu.cn

布鲁氏菌病是由布鲁氏菌引起的一种严重的慢性传染性疾病。（剩余12821字）