基于AIEgens增强的CRISPR/Cas12a荧光探针设计及在单核细胞增生李斯特菌快速检测中的应用  

AIEgens probes enhanced the sensitivity of CRISPR/Cas12a system for Listeria monocytogenes rapid detection

作　　者：叶莎玲 董香梅 郭宇乾 熊勇华[1,3] YE Shaling;DONG Xiangmei;GUO Yuqian;XIONG Yonghua(State Key Laboratory of Food Science and Resource,Nanchang University,Nanchang 330047,China;Fuzhou Jinyu Medical Laboratory Co.,Ltd.,Fuzhou 350001,China;Sino-German Joint Research Institute,Nanchang University,Nanchang 330047,China)

机构地区：[1]南昌大学食品科学与资源挖掘国家重点实验室,江西南昌330047 [2]福州金域医学检验实验室有限公司,福建福州350001 [3]南昌大学中德联合研究院,江西南昌330047

出　　处：《南昌大学学报（理科版）》2024年第3期251-260,共10页Journal of Nanchang University(Natural Science)

基　　金：国家重点实验室资助项目(20232BCD44004);江西省重点研发计划(20232BBG70030)。

摘　　要：研制了一种基于聚集诱导发光染料(aggregation-induced emission fluorogens,AIEgens)增强的荧光探针,建立了基于重组酶聚合酶扩增(recombinase polymerase amplification,RPA)结合CRISPR/Cas12a高灵敏检测单核细胞增生李斯特菌(Listeria monocytogenes,LM)的新方法。利用阳离子AIEgens与双链DNA结合荧光极大增强的特性,构建了一种含双链以及单链DNA的探针。该探针5’末端携带一个荧光淬灭基团,双链DNA片段结合了多个AIEgens;利用crRNA可准确识别靶标序列并激活Cas12a反式切割活性的特点,切割荧光探针单链DNA产生荧光信号,从而实现LM高灵敏检测。将AIEgens增强型荧光探针与CRISPR/Cas12a结合,在无扩增情况下检测DNA片段的检出限为0.37 pmol·L^(-1),灵敏度较传统荧光探针高40倍。将以上检测体系与RPA反应联用,检测LM的检出限为3×10^(1) CFU·mL^(-1);该方法检测人工污染LM的牛奶以及金针菇样本,批内以及批间平均回收率介于91.14%~108.47%,变异系数小于12.71%。构建了一种基于AIEgens增强的CRISPR/Cas荧光探针,极大地提高了基于荧光信号输出的CRISPR/Cas方法检测灵敏度,实现了食源性致病菌的高灵敏检测。An enhanced fluorescent DNA probe based on aggregation-induced emission fluorogens(AIEgens)was developed,and a new method for sensitive detection of Listeria monocytogenes(LM)was established based on recombinant polymerase amplification(RPA)combined with CRISPR/Cas12a.The AIEgens enhanced probe contained both single-stranded and double-stranded DNA sequence,where a Black Hole Quencher group was labeled on the 5’end of probe,and the cationic AIEgens dramatically enhanced the fluorescent intensity of DNA probe via binding with the dsDNA.The fluorescent signal of the developed probe was turned on by the trans-cleavage of Cas12a protein,which was trigged by the target DNA fragment of LM.Using the AIEgens enhanced probes as a signal output,the sensitivity of CRISPR/Cas12a for the dsDNA fragment was achieved at 0.37 pmol·L^(-1),40 times higher than that of traditional fluorescent reporter.Combined with recombinase polymerase amplification,the sensitivity of the proposed CRISPR/Cas12a system was achieved at 3×10^(1) CFU·mL^(-1) for the LM determination.Intra-and inter-assays were evaluated by determining the artificially contaminated LM milk and flammulina mushroom samples,and the average recoveries ranged from 91.14%to 108.47%with the coefficient of variation less than 12.71%,indicating an accepted accuracy and precision.An AIEgens enhanced fluorescent DNA probe was constructed to improve the detection efficiency of CRISPR/Cas system,the proposed CRISPR/Cas system achieved highly sensitivity for the foodborne pathogenic detection.

关 键 词：CRISPR/Cas12a 聚集诱导发光分子 单核细胞增生李斯特菌 食源性致病菌 快速检测 

分 类 号：R155.5[医药卫生—营养与食品卫生学] Q253[医药卫生—公共卫生与预防医学]