蜡样芽孢杆菌cytK和nhe毒素基因双重核酸试纸条快速检测方法的建立及其评价  

Establishment and evaluation of method for rapid detection of Bacillus cereus cytK and nhe toxin genes by dual nucleic acid test strips

作　　者：杨继飞 潘蓓珍 刘岩 周钰娇 杨建宇 张先宇 丁文博 李昊宇 孙丽媛 YANG Jifei;PAN Beizhen;LIU Yan;ZHOU Yujiao;YANG Jianyu;ZHANG Xianyu;DING Wenbo;LI Haoyu;SUN Liyuan(Department of Clinical Pathogen Laboratory,School of Medical Technology,Beihua University,Jilin 132013,China;Jilin Institute of Disease Control and Prevention,China Railway Shenyang Bureau Group Co.,Ltd,Jilin 132001,China)

机构地区：[1]北华大学医学技术学院临床病原学检验教研室,吉林吉林132013 [2]中国铁路沈阳局集团有限公司吉林疾病预防控制所,吉林吉林132001

出　　处：《吉林大学学报(医学版)》2025年第2期516-525,共10页Journal of Jilin University:Medicine Edition

基　　金：吉林省科技厅科技发展计划项目(20230204085YY,20230202059NC);北华大学研究生创新计划项目([2023] 070)。

摘　　要：目的:采用聚合酶链式反应(PCR)和胶体金技术建立1种蜡样芽孢杆菌cytK和nhe2种毒素基因双重核酸试纸条快速检测方法,并对其特异性、灵敏度、重复性和稳定性进行评价。方法:采用煮沸法提取蜡样芽孢杆菌DNA,以蜡样芽孢杆菌细胞毒素cytK和非溶血性肠毒素nhe为靶基因设计特异性引物,通过克隆转化鉴定PCR产物,确定胶体金标记链霉亲和素、质量控制线(C线)、cytK检测线(T_(1))和nhe检测线(T_(2))最佳标记量,组装核酸试纸条并对其特异性、灵敏度、重复性和稳定性进行评价。结果:蜡样芽孢杆菌DNA浓度为248 mg·L^(-1),纯度为1.8~2.0;经克隆转化和测序比对,PCR产物与GenBank数据库中已登记的蜡样芽孢杆菌cytK和nhe相似性均为100%。在pH为7.0的条件下,每200μL胶体金溶液中链霉亲和素最佳标记量为6.0μL;质量控制线(C线)最佳标记量为2.00 g·L^(-1),cytK基因检测线(T1)最佳标记量为0.550 g·L^(-1),nhe基因检测线(T_(2))最佳标记量为0.2 g·L^(-1)。特异性检测,仅蜡样芽孢杆菌组显示阳性结果,试纸条与金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌和枯草芽孢杆菌无交叉反应,与电泳结果一致;灵敏度检测,当双重核酸试纸条中蜡样芽孢杆菌DNA浓度降至10^(-2)mg·L^(-1)时,可观察到C线、T1线和T_(2)线3个条带,检测限为琼脂糖凝胶电泳(10^(-1)mg·L^(-1))的1/10;重复性检测,由不同实验室不同人员对试纸条进行验证,结果一致;稳定性检测,在第6、9和12个月进行试纸条稳定性验证,稳定性良好。结论:本研究建立的双重核酸试纸条法可以同时检测蜡样芽孢杆菌cytK和nhe毒素基因,灵敏度高,特异性强,可实现快速可视化检测。Objective:To establish a method of dual nueleic acid test strips for rapid detection of Bacillus cereus cytK and nhe toxin genes based on polymerase chain reaction(PCR)and colloidal gold technique,and to evaluate its specificity,sensitivity,repeatability and stability.Methods:Bacillus cereus DNA was extracted by boiling method.Specific primers were designed with Bacillus cereus cytK and nhe as the target genes.Clonal transformation was used to identify the PCR products.The optimal labeling amounts of colloidal gold-labeled streptavidin,quality control line(C line),cytK detection line(T_(1))and nhe detection line(T_(2))were determined.The nucleic acid test strips were assembled and its specificity,sensitivity,reproducibility and stability were evaluated.Results:The DNA concentration of Bacillus cereus was 248 mg·L^(-1),and the purities were 1.8-2.0.After cloning and plasmid sequencing,the similarities between the PCR products and the sequences of cytK and nhe registered in the GenBank database were 100%.Under the condition of pH 7.0,the optimal amount of streptavidin labeling per 200μL of colloidal gold solution was 6.0μL;the optimal marking amount was 2.00 g·L^(-1) for the quality control line(C line),0.550 g·L^(-1) for cytK gene detection line(T_(1))and 0.2 g·L^(-1) for nhe gene detection line(T_(2)).In the specificity test,positive result on the test strips was seen only for Bacillus cereus,and no cross-reactivity was observed for Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa and Bacillus subtilis,which were consistent with the electrophoresis results.Sensitivity assay showed that even when DNA concentration was reduced to 10-2 mg·L^(-1),three bands(C line,T_(1) line and T_(2) line)could be observed,and the detection limit of the test strip was one-tenth of agarose gel electrophoresis(10-1 mg·L^(-1)).The nucleic acid test strips were verified by different operators in different laboratories,and the results were consistent.The stability of the test strips was verified at the 6th,9th and 1

关 键 词：蜡样芽孢杆菌 cytK NHE 双重核酸试纸条 

分 类 号：R446.5[医药卫生—诊断学]