摘要
目的:为实现现制直饮品中铜绿假单胞菌的快速检测,开发基于酶促重组等温扩增(ERA)技术的荧光检测法和试纸条检测法。方法:以铜绿假单胞菌的弹性蛋白酶编码(las B)基因为靶标基因设计引物探针,建立两种基于ERA的快速检测方法(荧光检测法、试纸条检测法)。通过系统验证试验,对检测系统的特异性、灵敏度、实际样本适用性等性能指标进行全面评估。结果:所建方法对铜绿假单胞菌展现出良好的特异性和包容性。在灵敏度方面,对于目标DNA的检测下限分别达到10-1ng/μL和10-3ng/μL。人工污染结果显示,荧光检测法和试纸条检测法分别通过6 h和4 h短时前增菌,最低检出限可达1 CFU/mL。采用上述方法检测20份市售样品,结果显示:10份样品呈阳性,阳性符合率达50%。与国家标准GB8538-2022的比对分析显示准确率达100%,证实此方法在饮品基质中的稳定性和检测结果的准确性。结论:基于ERA技术构建的铜绿假单胞菌双模式快速检测方法(荧光检测法和试纸条检测法),于37℃体温下扩增20 min左右,即可获取检测结果。方法简单、准确、快速、灵敏,为现制直饮品中铜绿假单胞菌的快速检测提供了新的技术手段。
Objective:To achieve the rapid detection of Pseudomonas aeruginosa in ready-to-drink beverages,this study developed and rapid detection techniques utilizing enzymatic recombinase amplification(ERA).Methods:According to the elastase LasB(lasB)gene of Pseudomonas aeruginosa,primers and probes for ERA detection were designed and optimized.This method utilizes two ERA-based approaches:a fluorescence assay and a test strip assay,were established.Then,the specificity,sensitivity and applicability to commercially available samples were comprehensively evaluated through systematic validation experiments.Results:The experimental data indicate that the established method exhibits good specificity and inclusivity for Pseudomonas aeruginosa,demonstrating.The limit of detection(LOD)for target DNA was determined to be 10-1 ng/μL for the fluorescence assay and 10-3 ng/μL for the test strip assay,demonstrating good sensitivity.In artificially contaminated samples,the two assays achieved a detection limit of 1 CFU/mL after pre-enrichment times of 6 h and 4 h,respectively.When applied to 20 commercially available samples,10 samples tested positive,yielding a positive rate of 50%.Comparison with the national standard GB 8538-2022 demonstrated 100%accuracy,confirming the stability of the method in ready-to-drink beverages and the accuracy of the detection results.Conclusion:A dual-mode rapid detection method for Pseudomonas aeruginosa,utilizing ERA technology with both fluorescence and test strip assay formats,provides results in approximately 20 minutes of amplification at 37℃.This simple,accurate,rapid,and sensitive method offers a novel technical approach for the and rapid detection of Pseudomonas aeruginosa in ready-to-drink beverages.
作者
赵健淞
杨艳歌
刘通
魏莹
李红娜
李涛
孙冬梅
袁飞
ZHAO Jiansong;YANG Yange;LIU Tong;WEI Ying;LI Hongna;LI Tao;SUN Dongmei;YUAN Fei(College of Life Science and Biotechnology,Heilongjiang Bayi Agricultural University,Daqing 163000,Heilongjiang;Key Laboratory of Food Quality and Safety for State Market Regulation,Chinese Academy of Inspection and Quarantine,Beijing 100176)
出处
《中国食品学报》
北大核心
2025年第4期376-385,共10页
Journal of Chinese Institute Of Food Science and Technology
基金
国家重点研发计划项目(2022YFF1100900)
国家市场监督管理总局技术保障专项(2023YJ17)。
关键词
铜绿假单胞菌
酶促等温扩增
现制直饮品
快速检测
Pseudomonas aeruginosa
enzymatic recombinase amplification
ready-to-drink beverages
rapid detection
