实时荧光环介导等温扩增技术检测猪肉中的假单胞菌
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摘要
根据假单胞菌16S rDNA基因序列设计引物,并向反应体系中加入饱和型核酸染料Eva Green,利用实时荧光检测仪,建立假单胞菌属实时荧光环介导等温扩增技术(LAMP)。研究中对62株假单胞菌的16S rDNA基因通过DNAMAN软件进行序列比对后,针对共有片段设计扩增引物。对扩增体系进行了优化,优化结果经电泳鉴定。通过12株假单胞菌和23株非假单胞菌进行特异性验证,并比较了实时荧光LAMP与普通LAMP的灵敏度,对人工污染样品的检出限进行了测定。结果表明,特异性验证中12株假单胞菌为阳性,23株非假单胞菌为阴性。实时荧光LAMP检测纯菌的灵敏度为36 CFU/mL,是普通LAMP灵敏度的10倍。人工污染样品的检出限为1.73×10~3 CFU/g。本研究中建立了一种检测冷鲜猪肉中假单胞菌属的方法,实现了多个菌种的同时检测,避免了单一菌种检测的局限性,该方法快速、准确、灵敏度高,可在2 h内完成冷鲜猪肉中假单胞菌的检测。
In this work, a real-time fluorescence loop-mediated isothermal amplification(LAMP) assay was established to detect Pseudomonas in pork. Based on the published 16 S rDNA gene sequence, the 16 S rDNA genes of 62 strains of Pseudomonas were sequenced by DNAMAN software to obtain the consensus fragment, which was used to design primers. The saturated nucleic acid dye Eva Green was added to the reaction system, which allowed amplification products to be measured by real-time fluorescence platforms. The amplification system was optimized and the optimization results were identified by electrophoresis. The specificity and sensitivity was compared with ordinary LAMP,and the detection limit of artificially contaminated pork was determined. The results showed that 12 strains of Pseudomonas are positive in the specific verification and 23 strains of non-Pseudomonas are negative. The sensitivity for Pseudomonas is 36 CFU/mL in pure cultures, which is10 times more sensitive than the ordinary LAMP. The detection limit of artificially contaminated samples is 1.73×103 CFU/g. In this work, a method for detecting Pseudomonas in refrigerated pork was established, which can simultaneously detect Pseudomonas spp. and avoid the limitations of single species detection. The real-time fluorescent LAMP detection technology established in this work is less time-consuming,accurate and has high specificity and sensitivity. Pseudomonas in fresh pork could be detected within 2 hours by this method.
In this work, a real-time fluorescence loop-mediated isothermal amplification(LAMP) assay was established to detect Pseudomonas in pork. Based on the published 16 S rDNA gene sequence, the 16 S rDNA genes of 62 strains of Pseudomonas were sequenced by DNAMAN software to obtain the consensus fragment, which was used to design primers. The saturated nucleic acid dye Eva Green was added to the reaction system, which allowed amplification products to be measured by real-time fluorescence platforms. The amplification system was optimized and the optimization results were identified by electrophoresis. The specificity and sensitivity was compared with ordinary LAMP,and the detection limit of artificially contaminated pork was determined. The results showed that 12 strains of Pseudomonas are positive in the specific verification and 23 strains of non-Pseudomonas are negative. The sensitivity for Pseudomonas is 36 CFU/mL in pure cultures, which is10 times more sensitive than the ordinary LAMP. The detection limit of artificially contaminated samples is 1.73×103 CFU/g. In this work, a method for detecting Pseudomonas in refrigerated pork was established, which can simultaneously detect Pseudomonas spp. and avoid the limitations of single species detection. The real-time fluorescent LAMP detection technology established in this work is less time-consuming,accurate and has high specificity and sensitivity. Pseudomonas in fresh pork could be detected within 2 hours by this method.
引文
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[30]Ye L,Li Y,Zhao J,et al.Development of a real-time loop-mediated isothermal amplification assay for the sensitive and rapid detection of Listeria monocytogenes[J].Letters in Applied Microbiology,2015,61(1):85-90
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[32]王瑾,林丽萍,郜彦彦,等.实时荧光环介导等温扩增快速检测鸡肉中沙门氏菌[J].食品科学,2016,37(24):170-174WANG Jin,LIN Li-ping,GAO Yan-yan,et al.Development and application of a real-time loop-mediated isothermal amplification assay for rapid detection of Salmonella enterica ser.enteritis retrieved from chicken[J].Food Science,2016,37(24):170-174
[2]Stéphane Chaillou,Aurélie Chaulot-Talmon,Hélène Caekebeke,et al.Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage[J].Isme Journal,2015,9(5):1105-1118
[3]Lambert A D,Smith J P,Dodds K L.Shelf life extension and microbiological safety of fresh meat-a review[J].Food microbiology,1991,8(4):267-297
[4]王永锋.生鲜产品在途配送质量控制及可追溯系统关键技术研究[D].重庆:重庆大学,2012WANG Yong-feng.Research on key technologies about quality control of fresh product distribation in road and traceability system[D].Chongqing:Chongqing University,2012
[5]Guang-yu Wang,Hu-hu Wang,Yi-wei Han,et al.Evaluation of the spoilage potential of bacteria isolated from chilled chicken in vitro and in situ[J].Food Microbiology,2017,63:139-146
[6]Liang Rongrong,Yu Xiaoqiao,Wang Renhuan,et al.Bacterial diversity and spoilage-related microbiota associated with freshly prepared chicken products under aerobic conditions at 4℃[J].Journal of Food Protection,2012,75(6):1057-1062
[7]Pennacchia C,Ercolini D,Villani F.Spoilage-related microbiota associated with chilled beef stored in air or vacuum pack[J].Food Microbiology,2011,28(1):84-93
[8]Yang X,Zhu L,Zhang Y,et al.Microbial community dynamics analysis by high-throughput sequencing in chilled beef longissimus steaks packaged under modified atmospheres[J].Meat Science,2018,141:94-102
[9]Andreani,N A,Fasolato L.Pseudomonas and Related Genera[M].Cambridge:Woodhead Publltd,2017
[10]Chunxing Wang,Bin Zhang,Xiaomei Zhuang.Abiochemical system of rapidly detecting bacteria based on ATP bioluminescence technology[J].European Food Research and Technology,2013,236(1):41-46
[11]Leona Wunderlichová,Leona Buňková,Marek Koutny,et al.Novel touchdown-PCR method for the detection of putrescine producing Gram-negative bacteria in food products[J].Food Microbiology,2013,34(2):268-276
[12]Yamaguchi N,Ohba H,Nasu M.Simple detection of small amounts of Pseudomonas cells in milk by using a microfluidic device[J].Letters in Applied Microbiology,2010,43(6):631-636
[13]Abdalhai M H,Fernandes A M,Bashari M,et al.Rapid and sensitive detection of foodborne pathogenic bacteria(Staphylococcus aureus)using an electrochemical DNAgenomic biosensor and its application in fresh beef[J].Journal of Agricultural and Food Chemistry,2014,62(52):12659-12667
[14]Domesle K J,Yang Q,Hammack T S,et al.Validation of a Salmonella loop-mediated isothermal amplification assay in animal food[J].International Journal of Food Microbiology,2018,264:63-76
[15]Schrader C,Schielke A,Ellerbroek L,et al.PCRinhibitors-Occurrence,properties and removal[J].Journal of Applied Microbiology,2012,113(5):1014-1026
[16]LI Ping,WEN Ping-wei,XU Heng-yi,et al.Research progress in application of flow cytometry in detection of foodborne pathogen[J].Science&Technology of Food Industry,2013,34(14):375-379
[17]Yang Liju,Bashir Rashid.Electrical/electrochemical impedance for rapid detection of foodborne pathogenic bacteria[J].Biotechnology Advances,2008,26(2):135-150
[18]Zhang X,Lowe S B,Gooding J J.Brief review of monitoring methods for loop-mediated isothermal amplification(LAMP)[J].Biosensors&Bioelectronics,2014,61:491-499
[19]Pennacchia C,Ercolini D,Villani F.Development of a Real-Time PCR assay for the specific detection of Brochothrix thermosphacta in fresh and spoiled raw meat[J].International Journal of Food Microbiology,2009,134(3):230-236
[20]Han F,Ge B.Quantitative detection of Vibrio vulnificus in raw oysters by real-time loop-mediated isothermal amplification[J].International Journal of Food Microbiology,2010,142(1-2):60-66
[21]Kodogiannis V S,Pachidis T,Kontogianni E.An intelligent based decision support system for the detection of meat spoilage[J].Engineering Applications of Artificial Intelligence,2014,34:23-36
[22]Nidhi G,Colin H,Ross P R,et al.The prevalence and control of bacillus and related spore-forming bacteria in the dairy industry[J].Frontiers in Microbiology,2015,6
[23]Jones Rhys J.Observations on the succession dynamics of lactic acid bacteria populations in chill-stored vacuum-packaged beef[J].International Journal of Food Microbiology,2004,90(3):273-282
[24]Takano C,Seki M,Kim D W,et al.Development of a novel loop-mediated isothermal amplification method to detect guiana extended-spectrum(GES)beta-lactamase genes in Pseudomonas aeruginosa[J].Frontiers in Microbiology,2019,10
[25]Chen Z,Chai A,Li B,et al.New loop-mediated isothermal amplification primer set useful for detecting Pseudomonas syringae pv.Tomato and diagnosis of tomato bacterial spot disease,CN108411017-A[P/OL].
[26]Xin L,Zhang L W,Meng Z X,et al.Development of a novel loop-mediated isothermal amplification assay for the detection of lipolytic Pseudomonas fluorescens in raw cow milk from north China[J].Journal of Dairy Science,2017,100(10):7802-7811
[27]Zhou T,Wang G,Zhang J.Loop mediated isothermal amplification kit,useful for detecting Pseudomonas bacteria in large yellow croaker fish,comprises buffer solution,magnesium sulfate,deoxynucleotidetriphosphate solution and forward inner primer solution,CN102952884-A;CN102952884-B[P/OL].
[28]Mao Z J,Qiu Y Y,Zheng L,et al.Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker(Pseudosciaena crocea)[J].Journal of Microbiological Methods,2012,89(3):179-184
[29]贾雅菁,付博宇,王羽,等.实时荧光环介导等温扩增技术检测牛乳中的蜡样芽孢杆菌[J].食品科学,2016,37(6):184-189JIA Ya-jing,FU Bo-yu,WANG Yu,et al.Detection of Bacillus cereus in milk by real-time fluorescence loop-mediated isothermal amplification method[J].Food Science,2016,37(6):184-189
[30]Ye L,Li Y,Zhao J,et al.Development of a real-time loop-mediated isothermal amplification assay for the sensitive and rapid detection of Listeria monocytogenes[J].Letters in Applied Microbiology,2015,61(1):85-90
[31]Wang D.Novel primers for increased specificity and sensitivity for the detection of Staphylococcus aureus by real-time LAMP[J].Cyta-Journal of Food,2016,14(1):88-91
[32]王瑾,林丽萍,郜彦彦,等.实时荧光环介导等温扩增快速检测鸡肉中沙门氏菌[J].食品科学,2016,37(24):170-174WANG Jin,LIN Li-ping,GAO Yan-yan,et al.Development and application of a real-time loop-mediated isothermal amplification assay for rapid detection of Salmonella enterica ser.enteritis retrieved from chicken[J].Food Science,2016,37(24):170-174