食品与发酵工业 >

2020 , Vol. 46 >Issue 23: 198 - 206

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.024574

分析与检测

基于blaCARB-17基因建立水产品中副溶血弧菌的环介导等温扩增技术检测方法

  • 胡元庆 ,
  • 沈子晨 ,
  • 李凤霞 ,
  • 吕琳雪 ,
  • 周赞虎
展开
  • 1(闽南师范大学 生物科学与技术学院,福建 漳州,363000);
    2(扬州大学,江苏省人兽共患病学重点实验室,江苏 扬州,225000);
    3(漳州海关综合技术服务中心,福建 漳州,363000)
博士,副教授(通讯作者,E-mail:huyuanqing1979@163.com)

收稿日期: 2020-05-28

  修回日期: 2020-07-12

  网络出版日期: 2020-12-30

基金资助

福建省自然科学基金面上项目(2017J01453);江苏省人兽共患病学重点实验室资助项目(R1402)

收起

blaCARB-17-based LAMP assay for detectingVibrio parahaemolyticus in aquatic products

  • HU Yuanqing ,
  • SHEN Zichen ,
  • LI Fengxia ,
  • LYU Linxue ,
  • ZHOU Zanhu
Expand
  • 1(School of Biological Science and Biotechnology,Minnan Normal University,Zhangzhou 363000,China);
    2(Jiangsu Key Laboratory of Zoonosis,Yangzhou University,Yangzhou 225000,China);
    3(Comprehensive Technical Service Center,Zhangzhou Customs,Zhangzhou 363000,China)

Received date: 2020-05-28

  Revised date: 2020-07-12

  Online published: 2020-12-30

Fold

摘要

以β内酰胺酶编码基因-17(class A carbenicillin-hydrolyzing β-lactamase family-17,blaCARB-17)为靶标,建立一种检测水产品中副溶血弧菌的高特异性环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术。用Bacillus stearothermophilus(Bst) DNA聚合酶于63 ℃反应60 min,扩增副溶血弧菌ATCC17802的blaCARB-17基因,产物用质量浓度20 g/L琼脂糖凝胶电泳和SYBR Green I染色鉴定,优化体系的主要参数。用5株食源性病原菌作对照,验证体系的特异性;将基因组倍比稀释确定其灵敏度;稀释菌液涂抹新鲜虾样品做污染模拟实验,并对实际样品检测分析其可靠性。优化后体系中Mg2+最适浓度为2.4 mmol/L,dNTPs最佳反应浓度是0.96 mmol/L,Bst DNA聚合酶最适用量是4.8 U,内外引物浓度比是8:1,最佳反应条件是65 ℃、60 min。特异性实验显示仅副溶血弧菌为阳性;体系最低检测限为3.64×102ng/μL;模拟实验的最低检测限为10 CFU/mL;实际样品LAMP检测与PCR符合率100%。该研究建立的LAMP方法操作简便、特异性强、灵敏性高,可用于现场快速检测副溶血弧菌。

关键词： β内酰胺酶基因(blaCARB-17); 环介导等温扩增技术; 副溶血弧菌; 水产品; 快速检测

本文引用格式

胡元庆 , 沈子晨 , 李凤霞 , 吕琳雪 , 周赞虎 . 基于blaCARB-17基因建立水产品中副溶血弧菌的环介导等温扩增技术检测方法[J]. 食品与发酵工业, 2020 , 46(23) : 198 -206 . DOI: 10.13995/j.cnki.11-1802/ts.024574

Abstract

A highly specific loop-mediated isothermal amplification (LAMP) assay targeting theblaCARB-17 gene was developed for rapid and sensitive detection ofVibrio parahaemolyticus in aquatic products.The assay was optimized and conducted at 63 ℃ for 60 min usingBacillus stearothermophilus(Bst) DNA polymerase.Amplification was analyzed via 20 g/L agarose gel electrophoresis followed by SYBR Green Ⅰ staining.The specificity was determined by detectingV.parahaemolyticus ATCC 17802 and other 5 foodborne pathogens.The sensitivity was evaluated by detecting diluted genome DNA samples.The reliability was proved in both simulation experiment using experimentally contaminated shrimp samples and detection of aquatic samples from fish markets.The optimum detection condition was:2.4 mmol/L Mg2+,0.96 mmol/L dNTPs,4.8 UBst DNA polymerase,the ratio of inner and outer primer was 8:1,and react at 65 ℃ for 60 min.The result of specificity showed thatV.parahaemolyticus ATCC 17802 was positive,and other 5 control strains were negative.The detection limit of LAMP assay was 3.64×102 ng/μL,and the detection limit in the simulation experiment was 10 CFU/mL.The LAMP assay showed 100% consistency with conventional PCR for detecting practical samples.The LAMP assay established in this experiment is convenient,sensitive and specific,and is suitable for rapid in-field detection ofV.parahaemolyticus.

Key words： β-lactamase encoding gene (blaCARB-17); loop-mediated isothermal amplification; Vibrio parahaemolyticus; aquatic products; fast detection

参考文献

[1] YU Q Q,NIU M Y,YU M Q,et al.Prevalence and antimicrobial susceptibility ofVibrio parahaemolyticus isolated from retail shellfish in Shanghai[J].Food Control,2016,60:263-268.
[2] LAW J W F,AB MUTALIB N S,CHAN K G,et al.Rapid methods for the detection of foodborne bacterial pathogens:Principles,applications,advantages and limitations[J].Frontiers in Microbiology,2015,5:770.
[3] ELBASHIR S,PARVEEN S,SCHWARZ J,et al.Seafood pathogens and information on antimicrobial resistance:A review[J].Food Microbiology,2018,70:85-93.
[4] LI L Z,MENG H M,GU D,et al.Molecular mechanisms ofVibrio parahaemolyticus pathogenesis[J].Microbiological Research,2019,222:43-51.
[5] JIANG Y H,CHU Y B,XIE G S,et al.Antimicrobial resistance,virulence and genetic relationship ofVibrio parahaemolyticus in seafood from coasts of Bohai Sea and Yellow Sea,China[J].International Journal of Food Microbiology,2019,290:116-124.
[6] XIE T F,XU X K,WU Q P,et al.Prevalence,molecular characterization,and antibiotic susceptibility ofVibrio parahaemolyticus from ready-to-eat foods in China[J].Frontiers in Microbiology,2016,7:549.
[7] TAIWO M,BAKER-AUSTIN C,POWELL A,et al.Comparison oftoxR andtlh based PCR assays forVibrio parahaemolyticus[J].Food Control,2017,77:116-120.
[8] SIRIKHARIN R,TAENGCHAIYAPHUM S,SANGUANRUT P,et al.Characterization and PCR detection of binary,Pir-like toxins fromVibrio parahaemolyticus isolates that cause acute hepatopancreatic necrosis disease (AHPND) in shrimp[J].PloS One,2015,10(5):e0126987.
[9] KIM S H,LEE Y S,KWAK H S.Rapid real-time PCR for the detection ofVibrio parahaemolyticus in seafood[J].International Journal of Infectious Diseases,2016,53:69.
[10] XU D S,JI L,WU X F,et al.Detection and differentiation ofVibrio parahaemolyticus by multiplexed real-time PCR[J].Canadian Journal of Microbiology,2018,64(11):809-815.
[11] ANAND P S S,SOBHANA K S,GEORGE K C,et al.Selection of specific cell wall antigen for rapid detection of fish pathogenicVibrio parahaemolyticus by enzyme immunoassay[J].Indian Journal Fish,2016,63(2):76-85.
[12] TASRIP N A,KHAIRIL MOKHTAR N F,HANAPI U K,et al.Loop mediated isothermal amplification;a review on its application and strategy in animal species authentication of meat based food products[J].International Food Research Journal,2019,26(1):1-10.
[13] IZUMIYA H,MORITA M,ARAKAWA E,et al.Development of a loop-mediated isothermal amplification assay forVibrio cholerae O1 and O139[J].Molecular and Cellular Probes,2019,45:65-67.
[14] YAMAZAKI W,ISHIBASHI M,KAWAHARA R,et al.Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection ofVibrio parahaemolyticus[J].BMC Microbiology,2008,8(1):163.
[15] CHEN S Y,GE B L.Development of atoxR-based loop-mediated isothermal amplification assay for detectingVibrio parahaemolyticus[J].BMC Microbiology,2010,10(1):41.
[16] YAMAZAKI W,KUMEDA Y,UEMURA R,et al.Evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection ofVibrio parahaemolyticus in naturally contaminated seafood samples[J].Food Microbiology,2011,28(6):1 238-1 241.
[17] LIU N W,ZOU D Y,DONG D R,et al.Development of a multiplex loop-mediated isothermal amplification method for thesimultaneous detection ofSalmonella spp.andVibrio parahaemolyticus[J].Scientific Reports,2017,7(1):115-129.
[18] WANG L,SHI L,SU J Y,et al.Detection ofVibrio parahaemolyticus in food samples using in situ loop-mediated isothermal amplification method[J].Gene,2013,515(2):421-425.
[19] WANG Y,LI D X,WANG Y,et al.Rapid and sensitive detection ofVibrio parahaemolyticus andVibrio vulnificus by multiple endonuclease restriction real-time loop-mediated isothermal amplification technique[J].Molecules,2016,21(1):111.
[20] PROMPAMORN P,SITHIGORNGUL P,RUKPRATANPORN S,et al.The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection ofVibrio parahaemolyticus[J].Letters in Applied Microbiology,2011,52(4):344-351.
[21] CHEN C,LIU P,ZHAO X,et al.A self-contained microfluidic in-gel loop-mediated isothermal amplification for multiplexed pathogen detection[J].Sensors and Actuators B:Chemical,2017,239:1-8.
[22] 胡元庆,黄玉萍,李凤霞,等.水产品中副溶血性弧菌LAMP检测方法的优化[J].现代食品科技,2017,33(6):313-320;247.
HU Y Q,HUANG Y P,LI F X,et al.Optimization of loop-mediated isothermal amplification methods for the detection ofVibrio parahaemolyticus in aquatic products[J].Modern Food Science and Technology,2017,33(6):313-320;247.
[23] 纪懿芳,胡文忠,姜爱丽,等.LAMP技术快速检测海产品副溶血弧菌的条件优化[J].食品工业科技,2015,36(20):59-63;67.
JI Y F,HU W Z,JIANG A L,et al.Optimize of loop-mediated isothermal amplification technology in detection ofVibrio parahaemolyticus[J].Science and Technology of Food Industry,2015,36(20):59-63;67.
[24] 韩小龙,张海燕,曹明秀,等.基于环介导等温扩增法(LAMP)检测上海市售贝类产品中副溶血性弧菌的毒力菌株[J].现代食品科技,2015,31(9):278-283.
HAN X L,ZHANG H Y,CAO M X,et al.Detection of virulentVibrio parahaemolyticus strains from commercial shellfish in Shanghai by loop-mediated isothermal amplification[J].Modern Food Science and Technology,2015,31(9):278-283.
[25] 周顺,高志鑫,张敏.创伤弧菌和副溶血弧菌双重LAMP检测方法的建立及初步应用[J].中国兽医学报,2016,36(11):1 875-1 881.
ZHOU S,GAO Z X,ZHANG M.The development and application of a duplex loop-mediated isothermal amplification method for rapid detection ofVibrio vulnificus andVibrio parahaemolyticus[J].Chinese Journal of Veterinary Science,2016,36(11):1 875-1 881.
[26] 相兴伟,郑斌,顾丽霞,等.双重LAMP技术快速检测水产品中副溶血性弧菌和霍乱弧菌的方法学研究[J].现代食品科技,2017,33(1):253-260.
XIANG X W,ZHENG B,GU L X,et al.Study on simultaneous detection ofVibrio Parahaemolyticus andVibrio Cholerae in aquatic products by loop-mediated isothermal amplification method[J].Modern Food Science and Technology,2017,33(1):253-260.
[27] CHIOU J C,LI R,CHEN S.CARB-17 Family of β-Lactamases mediates intrinsic resistance to penicillins inVibrio parahaemolyticus[J].Antimicrobial Agents &Chemotherapy,2015,59(6):3 593-3 595.
[28] LI R C,CHIOU J,CHAN E W C,et al.A novel PCR-based approach for accurate identification ofVibrio parahaemolyticus[J].Frontiers in Microbiology,2016,7:44.
[29] 纪懿芳,胡文忠,姜爱丽,等.海产品中副溶血弧菌的LAMP-HNB快速检测技术[J].食品与发酵工业,2015,41(7):142-148.
JI Y F,HU W Z,JIANG A L,et al.Rapid diagnostic methods forVibrio parahaemolyticus in seafood using LAMP-HNB[J].Food and Fermentation Industries,2015,41(7):142-148.
[30] 付世骞,曲艳艳,冯晓涵,等.环介导等温扩增-无电加热法检测乳中阪崎克罗诺杆菌[J].食品与发酵工业,2018,44(3):220-225.
FU S Q,QU Y Y,FENG X H,et al.Detection ofCronobacter sakazakii in milk by loop-mediated isothermal amplification without electric heating method[J].Food and Fermentation Industries,2018,44(3):220-225.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司