双重核酸纸条快速检测单核细胞增生李斯特菌的prfA和hly毒素基因  

A rapid method for detecting prfA and hly toxin genes of Listeria monocytogenes using double nucleic acid colloidal gold strips

作　　者：刘岩 杨建宇 周钰娇 丁文博 张先宇 高林然 潘蓓珍 杨继飞 赵云冬 LIU Yan;YANG Jianyu;ZHOU Yujiao;DING Wenbo;ZHANG Xianyu;GAO Linran;PAN Beizhen;YANG Jifei;ZHAO Yundong(Department of Clinical Biochemistry and Molecular Biology,School of Medical Technology,Beihua University,Jilin 132013,China)

机构地区：[1]北华大学医学技术学院临床生化及分子生物学检验教研室,吉林吉林132013

出　　处：《南方医科大学学报》2025年第2期387-394,共8页Journal of Southern Medical University

基　　金：吉林省科技发展计划项目(20230202059NC);吉林省科技厅科技发展计划项目(20230204085YY);北华大学研究生创新计划项目(研创合字【2023】022)。

摘　　要：目的基于聚合酶链反应和胶体金技术相结合的双重核酸试纸条方法快速检测单核细胞增生李斯特菌prfA和hly两种毒素基因。方法采用加热煮沸法提取单核细胞增生李斯特菌DNA,以单核细胞增生李斯特菌的prfA和hly为靶基因,分别用6-FAM标记prfA上游引物5'端,Biotin标记prfA下游引物5'端,Digoxin标记hly上游引物5'端和Biotin标记hly下游引物5'端,建立单核细胞增生李斯特菌的毒素基因检测方法,通过克隆转化、测序分析,克隆阳性对照品,研发试剂盒并对试剂盒特异性、灵敏度、复现性及稳定性进行评价,并通过样品检测对试剂盒进行验证。结果水煮法提取的单核细胞增生李斯特菌浓度为148.81±0.97ng/μL,A_(260)/A_(280)在1.8~2.0范围内;PCR产物经克隆转化、测序对比,与GenBank数据库中基因序列的同源性均为100%;特异性检测,仅单核细胞增生李斯特菌为阳性结果,与金黄色葡萄球菌、大肠埃希菌、蜡样芽胞杆菌无交叉反应;灵敏度检测,最低检测限为10^(-2)ng/μL,比琼脂糖凝胶电泳高10倍;复现性检测,不同实验室不同人员分别对核酸试纸条进行验证,结果一致;稳定性检测,在第6、9、12月对核酸试纸条进行验证,稳定性较好。样品检测结果显示,本试剂盒可准确快速的检出样品中单核细胞增生李斯特菌。结论本剂盒可同时检测单核细胞增生李斯特菌的prfA和hly毒素基因,具有特异性强、灵敏度高、复现性好、稳定性好等优点,对保障食品安全具有现实意义。Objective To detect prfA and hly toxin genes of Listeria monocytogenes using polymerase chain reaction(PCR)and colloidal gold technology.Methods L.monocytogenes DNA was extracted by boiling method.With prfA and hly of L.monocytogenes as the target genes,the 5'ends of upstream and downstream primers of prfA gene were labeled with 6-FAM and biotin,and the 5'ends of upstream and downstream primers of hly gene were labeled with digoxin and biotin,respectively,to establish the toxin gene detection method.Using cloning transformation,sequencing analysis,cloning of positive control products,the detection kid was developed and its specificity,sensitivity,reproducibility and stability were tested,followed by verification with sample testing.Results The concentration of L.monocytogenes DNA extracted by boiling method was 148.81±0.97 ng/μL,and the A_(260)/A_(280) ratio ranged from 1.8 to 2.0.The PCR products showed a 100% homology with the gene sequences in GenBank database after cloning,transformation and sequencing.The colloidal gold strip yielded positive results only for L.monocytogenes samples without cross-reactions with Staphylococcus aureus,Escherichia coli or Bacillus cereus,and its minimum detection limit was 10^(-2) ng/μL,demonstrating a 10-fold greater sensitivity of the test than agarose gel electrophoresis.The test also showed good reproducibility of the results when performed by different operators with good stability of the test strips after storage for 6 to 12 months.The test results showed that this kit could accurately and quickly detect L.monocytogenes in the test samples.Conclusion The detection kit developed in this study can simultaneously detect prfA and hly toxin genes of L.monocytogenes with good specificity,sensitivity,reproducibility and stability for use in food safety inspection.

关 键 词：单核细胞增生李斯特菌 prfA hly 双重核酸试纸条 

分 类 号：J62[艺术—音乐]