沙门菌荧光重组酶介导等温扩增检测方法的应用研究
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摘要
目的建立基于重组酶介导等温扩增(recombinase-aided amplification, RAA)快速、灵敏、特异检测沙门菌的方法。方法针对沙门菌invA基因保守区段设计特异性引物、探针,通过引物、探针筛选以及反应条件优化,建立检测沙门菌的荧光RAA方法,通过优化引物浓度、探针浓度、醋酸镁浓度、上样量来确定最佳反应体系组成。采用细菌纯培养物评估该方法的最低检测限、特异性等性能,通过模拟样品以及19份真实样品检测,同时采用国标法GB 4789.4-2016同步验证复核,评估该检测方法的可靠性。结果该方法在39℃条件下20 min内可完成检测反应。利用该方法能够特异检测沙门菌,不能检出其他肠道致病菌。该方法检测细菌纯培养物的灵敏度为19 cfu/mL,检测模拟样品的灵敏度为190 cfu/mL。采用建立的沙门菌荧光RAA方法对采集的环境样品、食品污染及人体肛拭子样品共计19份进行检测,按照GB 4789.4-2016方法同步检测,4份阳性,其中3份样本荧光RAA检测呈现沙门菌阳性,1份肛拭子样本该方法未能检出。结论荧光重组酶介导等温扩增法检测沙门菌快速、特异、简便、灵敏,能够在食源性疫情现场调查以及食源性疾病监测中发挥一定的作用。
Objective To establish a fluorescent recombinase-aided amplification(RAA) method for the rapid detectionof Salmonella. Methods Specific primers and probes were designed for the conservative region of Salmonella invA gene. Afluorescent RAA method for detecting Salmonella was established by screening primers and probes and optimizing reactionconditions.The RAA reaction conditions were optimized, including the concentration of primer, probe, Mg2+and DNA template.The minimum detection limit and specificity of this system were evaluated by using pure bacterial cultures. The reliability ofthe method was evaluated by the verification of simulated samples and 19 real samples with the verification of the nationalstandard method GB 4789.4-2016 as standard. Results Under the condition of 39 ℃, the verification can be completedwithin 20 min.This method can detect Salmonella specifically, but it cannot detect other enteric pathogens. The sensitivity ofthe method for detecting pure bacterial culture was 19 cfu/mL, and the sensitivity for detecting simulated sample was 190 cfu/m L. A total of 19 samples of environmental samples, food contamination and human anal swabs were detected by theestablished Salmonella fluorescent RAA method, which also were simultaneously detected according to GB 4789.4-2016 method. Four samples were positive, three of them were Salmonella positive and one anal swab was not detected byfluorescence RAA. Conclusions The fluorescent RAA method for the detection of Salmonella is rapid, specific, easy-to-use, and sensitive, which can play a role in foodborne epidemic situation investigation and foodborne disease surveillance.
Objective To establish a fluorescent recombinase-aided amplification(RAA) method for the rapid detectionof Salmonella. Methods Specific primers and probes were designed for the conservative region of Salmonella invA gene. Afluorescent RAA method for detecting Salmonella was established by screening primers and probes and optimizing reactionconditions.The RAA reaction conditions were optimized, including the concentration of primer, probe, Mg2+and DNA template.The minimum detection limit and specificity of this system were evaluated by using pure bacterial cultures. The reliability ofthe method was evaluated by the verification of simulated samples and 19 real samples with the verification of the nationalstandard method GB 4789.4-2016 as standard. Results Under the condition of 39 ℃, the verification can be completedwithin 20 min.This method can detect Salmonella specifically, but it cannot detect other enteric pathogens. The sensitivity ofthe method for detecting pure bacterial culture was 19 cfu/mL, and the sensitivity for detecting simulated sample was 190 cfu/m L. A total of 19 samples of environmental samples, food contamination and human anal swabs were detected by theestablished Salmonella fluorescent RAA method, which also were simultaneously detected according to GB 4789.4-2016 method. Four samples were positive, three of them were Salmonella positive and one anal swab was not detected byfluorescence RAA. Conclusions The fluorescent RAA method for the detection of Salmonella is rapid, specific, easy-to-use, and sensitive, which can play a role in foodborne epidemic situation investigation and foodborne disease surveillance.
引文
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[11]YANT F,LIX N,WANG L,et al.Development of a reverse transcription recombinase-aided amplification assay for the detection of coxsackievirus A10 and coxsackievirus A6 RNA[J].Arch Virol,2018,163(6):1455-1461.
[12]赵松,李婷,杨坤,等.重组酶介导的日本血吸虫特异性基因片段核酸等温扩增检测方法的建立[J].中国血吸虫病防治杂志,2018,30(3):273-277,306.2018-10-25
[2]JACKSON E E,ERTEN E S,MADDI N,et al.Detection and enumeration of four foodborne pathogens in raw commingled silo milk in the United States[J].JournalofFoodProtection,2012,75(8):1382-1393.
[3]KUMAR S,BALAKRISHNA K,BATRA H V.Enrichment-ELISAfor detection of Salmonella typhi from food and water samples[J].Biomed EnvironSci,2008,21(2):137-143.
[4]彭海滨.胶体金免疫层析法和荧光定量PCR联合检测沙门氏菌[D].福州:福建农林大学,2007.
[5]MOREIRAA N,CONCEI??OF R,CONCEI??OR D E C,et al.Detection of Salmonella typhimurium in raw meats using in-house prepared monoclonal antibody coated magnetic beads and PCR assay of the fimA gene[J].J Immunoassay Immunochem,2008,29(1):58-69.
[6]吕蓓,程海荣,严庆丰,等.用重组酶介导扩增技术快速扩增核酸[J].中国科学:生命科学,2010,40(10):983-988.
[7]杜雄伟,李叶,冮洁,等.肉制品中沙门氏菌invA基因实时荧光定量PCR检测方法的建立[J].食品工业科技,2013,34(12):68-70,80.
[8]吴家林,沙丹,马广源,等.沙门氏菌LAMP检测方法的建立[J].中国病原生物学杂志,2015,10(7):611-614.
[9]许会会,雷连成,谢芳,等.沙门氏菌PCR检测方法的建立[J].中国畜牧兽医,2010,37(4):94-97.
[10]钟伟军,赵明秋,邓中平,等.荧光定量PCR快速检测食品中沙门氏菌方法的建立及初步应用[J].中国预防兽医学报,2008,30(3):220-224.
[11]YANT F,LIX N,WANG L,et al.Development of a reverse transcription recombinase-aided amplification assay for the detection of coxsackievirus A10 and coxsackievirus A6 RNA[J].Arch Virol,2018,163(6):1455-1461.
[12]赵松,李婷,杨坤,等.重组酶介导的日本血吸虫特异性基因片段核酸等温扩增检测方法的建立[J].中国血吸虫病防治杂志,2018,30(3):273-277,306.2018-10-25