结合CRISPR/Cas12b与LAMP技术的乳粉中克罗诺杆菌属的检测

Detection of Cronobacter spp in milk powder by CRISPR/Cas12b and LAMP

将CRISPR/Cas12b与环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)方法相结合,应用于乳粉中克罗诺杆菌属的检测。通过基因组比较寻找克罗诺杆菌菌株基因内部的候选保守序列,针对克罗诺杆菌属特异性关键基因序列设计LAMP引物和向导RNA靶向序列,对其进行条件优化、特异性检测,并用人工污染样品对体系的灵敏度、重复性进行验证。结果表明该方法具有良好的特异性,方法的灵敏度达到了0.1 pg/μL;人工污染样品可检测到初始菌量<10 CFU/g的样品,方法检出限为<10 CFU/100 g。将该方法应用于51份市售乳粉样品以及52份模拟生产环境样品的检测,结果与传统国标方法一致,并且大幅缩短了实验时间。该方法可用于乳粉中克罗诺杆菌属的快速检测。

This study combined CRISPR/Cas12b with LAMP for the detection of Cronobacter spp in milk powder.Through genome comparison,candidate conserved sequence within the genes of Cronobacter spp were found.LAMP primers and guide RNAs were designed and synthesized based on the specific gene sequence of Cronobacter spp.The conditions and specificity detection were optimized,and the sensitivity and reproducibility of the system were verified with artificially contaminated samples.The results showed a high specificity and sensitivity of this method-the sensitivity reached 0.1 pg/μL;samples with initial bacterial counting less than 10 CFU/g could be detected;The detection limit was less than 10 CFU/100 g.51 commercial milk powder samples and 52 simulated production environment samples were later tested by using our established method,and the results were consistent with traditional national standard methods,significantly shortening the experimental time.In conclusion,this method could be used for rapid detection of Cronobacter spp in milk powder..