重组酶介导的等温扩增结合CRISPR/Cas12a检测志贺氏菌和克罗诺杆菌

Detection of Shigella and Cronobacter using recombinase-aided amplification combined with CRISPR/Cas12a

中文关键词:  志贺氏菌  克罗诺杆菌  重组酶介导的等温扩增  成簇规则的间隔短回文重复序列

英文关键词:Shigella  Cronobacter  recombinase-aided amplification  clustered regularly interspaced short palindromic repeats

中文摘要:

      目的 应用重组酶介导的等温扩增技术(recombinase-aided amplification, RAA)结合成簇规则的间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR), 建立志贺氏菌和克罗诺杆菌的快速检测方法。方法 通过筛选志贺氏菌和克罗诺杆菌特异性基因, 设计特异扩增引物、CRISPR脱氧核糖核酸(CRISPR deoxyribonucleic acid, crDNA)和探针, 将靶标进行RAA扩增后, 建立CRISPR检测方法。对方法的灵敏度、特异性和应用性进行评价, 并与国标检测方法进行对比。结果 本研究对志贺氏菌和克罗诺杆菌纯菌液DNA最低检出限分别为4.9×103 CFU/mL、4.4×104 CFU/mL, 基因组DNA最低检出限为6.8×10?3 ng/μL、5.7×10?3 ng/μL, 特异性好, 与其他病原菌无交叉反应。同时, 该方法成功检测出加标猪肉样品和人工污染奶粉中的志贺氏菌和克罗诺杆菌, 比国家标准检出限更低。结论 本研究建立的RAA-CRISPR方法可有效应用于志贺氏菌和克罗诺杆菌的检测, 这为快速筛查食品中是否污染这两种病原菌提供重要价值。

英文摘要:

      Objective To establish a rapid detection method for Shigella and Cronobacter using recombinase-aided amplification (RAA) combined with clustered regularly interspaced short palindromic repeats (CRISPR). Methods By selecting specific genes for Shigella and Cronobacter, specific amplification primers, CRISPR Deoxyribonucleic acid (crDNA), and probes were designed. After amplifying the target used RAA, a CRISPR detection method was established. The sensitivity, specificity, and applicability of the method were evaluated and compared with national standard detection methods. Results This research exhibited a detection limit of 4.9×103 CFU/mL for Shigella and 4.4×104 CFU/mL for Cronobacter in pure bacterial liquid DNA, with genomic DNA detection limits of 6.8×10?3 ng/μL and 5.7×10?3 ng/μL, respectively. It displayed excellent specificity with no cross-reactivity observed with other pathogens. Moreover, the method successfully detected Shigella and Cronobacter in spiked pork samples and artificially contaminated powdered milk at lower limits of detection compared to the national standard method. Conclusion The RAA-CRISPR method establish in this study can be effectively apply for the detection of Shigella and Cronobacter, providing significant value for the rapid screening of food contamination by these two pathogens.