[1]李同祥a,黄天姿a,孙会刚a,等.食源性致病菌生物芯片-RCA检测新方法[J].徐州工程学院学报(自然科学版),2019,(2):26-34.
 LI Tongxianga,HUANG Tianzia,SUN Huiganga,et al.A New Method for Detecting Food-borne Pathogenic Bacteria by Biochip-RCA[J].Journal of Xuzhou Institute of Technology(Natural Sciences Edition),2019,(2):26-34.
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食源性致病菌生物芯片-RCA检测新方法()
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《徐州工程学院学报》(自然科学版)[ISSN:1674-358X/CN:32-1789/N]

卷:
期数:
2019年第2期
页码:
26-34
栏目:
教授论坛
出版日期:
2019-05-30

文章信息/Info

Title:
A New Method for Detecting Food-borne Pathogenic Bacteria by Biochip-RCA
文章编号:
1674-358X(2019)02-0026-09
作者:
李同祥a黄天姿a孙会刚a汤 薇a田 林b李 文a张传丽a王春燕a
(徐州工程学院 a.食品(生物)工程学院; b.化学化工学院,江苏 徐州 221018)
Author(s):
LI TongxiangaHUANG TianziaSUN HuigangaTANG WeiaTIAN Linb LI WenaZHANG ChuanliaWANG Chunyana
(a.School of Food(Biology)Engineering; b.School of Chemical Engineering and Technology, Xuzhou Institute of Technology,Xuzhou 221018,China)
关键词:
生物芯片 滚环扩增 致病菌 检测探针
Keywords:
biochip rolling circle amplification(RCA) pathogenic bacteria detect probe
分类号:
Q789
文献标志码:
A
摘要:
将生物芯片技术和滚环扩增(rolling circle amplification,RCA)技术结合,建立一种在片RCA检测食源致病性菌新方法.依据待检测致病菌的基因组序列设计捕获探针(capture probe,CP)、检测探针(detect probe,DP)和滚环探针(rolling circle probe,RCP),其中捕获探针、检测探针与待检测致病菌基因组序互补,RCP不含任何待检菌基因组DNA序列,CP的5'末端修饰氨基,将其点样制备成CP微阵列,DP、RCP与待检菌gDNA混合后变性,将其与CP微阵列杂交并连接, RCA扩增反应体系中加入链亲和素进行在片扩增反应,分析检测结果.研究表明,该方法能灵敏、特异性地检测单一和混合靶标分子,以金黄色葡萄球菌为检测对象,其最低检测限可到达50 fg/μL,其线性范围是400 ~50 fg/μL,R2达到0.97,证实了该方法的可行性和可靠性,为食源性致病菌检测提供了新的技术方法.
Abstract:
A novel method for detecting food-borne pathogenic bacteria was developed by combining biochip with rolling circle amplification(RCA)technology.The capture probes(CP),detection probe(DP)and rolling circle probe(RCP)were designed according to the genome sequence of the pathogenic bacteria to be detected.CPs and DPs were designed to match sequences of the "target" pathogenic bacteria,but RCP does not contain any sequence of it and then chemically synthesized.The 5'end of CP is modified with amino groups.The CP microarray was fabricated.DP and RCP were denatured after mixing with gDNA of the "target" bacteria.They were hybridized with the CP microarray and performed ligation reactions.Streptavidin was added to the RCA amplification reaction system to perform RCA on chip.The chip was scanned and the results were analyzed.The results show that this method can detect single and mixed "target" molecules with high sensitivity and specificity.It can quantitatively detect the gDNA of pathogenic bacteria.The minimum detection limit can reach 50 fg/μL.Its linear range is 400~50 fg/μL and R2 reaches 0.97.The feasibility and reliability of this method was confirmed.The established method has the characteristics of high sensitivity,high signal-to-noise ratio,good specificity and no dependence on antibodies.It provides a new method and development direction for the detection of foodborne pathogenic bacteria.

参考文献/References:

[1] J NALLUR G,LUO C H,FANG L H,et al.Signal amplification by rolling circle amplification on DNA microarrays[J].Nucleic Acids Research,2001,29(23):118.
[2] 闻一鸣,李志清,童吉宇,等.免疫磁珠富集技术联合选择性培养基快速检测单增李斯特菌[J].生物工程学报,2013,29(5):672-680.
[3] TURNER K M,RESTAINO L,FRAMPTON E W.Efficacy ofchromocult coliform agar for coliform and escherichia coil detection in foods[J].Journal of Food Protection,2000,63(4):539-541.
[4] CHEN J,TANG J N,BHUNIA A K,et al.Development of a multi-pathogen enrichment broth for simultaneous growth of five common food-borne pathogens[J].Journal of General and Applied Microbiology,2015,61(6):224-231.
[5] LAW J W,ABMUTALIB N S,CHAN K G,et al.An insight into the isolation,enumeration,and molecular detection of listeriamonocytogenesin food[J].Frontiers in Microbiology,2015,6(114):1-15.
[6] YU Y G,WU H,LIU YY,et al.A multipathogen selective enrichment broth for simultaneous growth of salmonella enterica serovar enteritis,staphylococcus aureus,and listeria monocytogenes[J].Canadian Journal of Microbiology,2010,56(7):585-597.
[7] GARDLNIA F,LANCIOTTIB R,SINIGAGLIAB M,et a1.A head space gas chromatographic approach for the monitoring of the microbial cell activity and the cell viability evaluation[J].Journal of Microbio-logical Methods,1997,29(2):103-114.
[8] PREVITE J J.Radiometric detection of some food-borne bacteria[J].Applied and Environmental Microbiology,1972,24(4):535-539.
[9] WANG W H,ZhANG X W,ZHOU J B,et al.Research progress on fast detection methods of foodborne pathogenic microbe[J].J Food Safe Qual,2010,27(4):182-188.
[10] TERAMURA H,SEKIGUCHI J,INOUE K.A novel chromogenic screening medium for isolation ofenterohemorrhagic Escherichia coli[J].Biocontrol Sci,2013,18(2):111-115.
[11] RITTER V,KIRCHER S,STURM K,et al.Evaluation of BD BBL CHROM agar staph aureus medium using AOAC and ISO culture methods.performance tested method 100503[J].J AOAC Int,2009,92(5):1432-1453.
[12] RITTER V,KIRCHER S,DICK N.USDA FSIS,FDA BAM,and ISO culture methods BD BBL CHROMagar O157 media[J].J Aoac Int,2009,92(4):1118-1127.
[13] BEBESH D L,CROWLY E S,BIRD P M.3MPetrifilm environmental listeria plate[J].J AOAC Int,2013,96(2):225-228.
[14] SLIVA B O,CARAVIELLO D Z,RODRIGUES A C,et al.evaluation of petrifilm for the isolation of staphylococcus aureus from milk samples[J].J Dairy Sci,2005,88(8):3000-3008.
[15] KODAKA H,GOTO K,UMEKI K,et al.Comparative evaluation of the desoxycholate agar nissui food stamp and swab methods for estimating coliform organisms in poultry processing plants after cleaning in Japan[J].J Basic Microbiol,2004,44(6):445-450.
[16] YAMAZAKI W,KUMEDA Y,UEMURA R,et al.Evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of vibrioparahaemolyticus in naturally contaminated seafood samples[J].Food Microbiol,2011,28(6):1238-1241.
[17] NIESSEN L,LUO J,DENSCHLAG C,et al.The application of loop-mediated isothermal amplification(LAMP)in food testing for bacterial pathogens and fungal contaminants[J].Food Microbiol,2013,36(2):191-206.
[18] KUMAR R,SURENDRAN P K,THAMPURAN N.Rapid quantification of Salmonella in seafood by real-time PCR assay[J].J Microbiol Biotechnol,2010,20(3):569-573.
[19] 王娉,杨海荣,袁飞,等.多重实时荧光定量聚合酶链反应检测携带耐药基因及肠毒素 A 基因的金黄色葡萄球菌[J].疾病监测,2011,26(12):999-1003.
[20] XIA Q F,XU S X,WANG D S,et al.Development of a novel quantitative real-time assay using duplex scorpion primer for detection of chlamydia trachomatis[J].Exp Mol Pathol,2007,83(1):119-124.
[21] TSAIG J,COUSIN M A.Enzyme-Linked immunosorbent assay for detection of molds in cheese and yogurt[J].J Dairy Sci,1990,73(12):3366-3378.
[22] BROOKS B W,LUTZE-WALLACE C L,DEVENISH J.Comparison of an antigen-capture enzyme-linked immunosorbent assay with bacterial culture for detection of salmonellain poultry-hatchery environmental samples[J].Canadian Journal of Veterinary Research,2014,78(1):68-71.
[23] TU Z,CHEN Q,LI Y,et al.Identification and characterization of species-specificnanobodies for the detection of Listeria monocytogenes in milk[J].Analytical Biochemistry,2016,15(493):1-7.
[24] NAAS T,CUZON G,TRUONG H,et al.Evaluation of a DNA microarray,the check-points ESBS / KPC array,for rapid detection of TEM.SHV,and CTX-M extended-spectrum beta-lactamases and KPC carbapenemases[J].Antimicrob Agents Chemother,2010,54(8):3086-3092.
[25] BATCHELOR M,HOPKINS K L,LIEBANA E,et al.Development of a miniaturized microarray for the rapid identification of antimicrobial resistance genes in gram-negative bacteria[J].Int J Antimicrob Agents,2008,31(5):440-451.
[26] YOON J Y,KIM B.Lab-on-a-chip pathogen sensors for food safety[J].Sensors(Basel),2012,12(8):10713-10741.
[27] GANZ K,GILL A.Inhibition of polymerase chain reaction for the detection of escherichia coli O157:H7 and salmonellaenterica on walnut kernels[J].Food Microbiol,2013,35(1):15-20.
[28] SUO B,HE Y,PAOLI G,et al.Development of an oligonucleotide-based microarray to detect multiple foodborne pathogens[J].Molecular and Cellular Probes,2010,24(2):77-86.
[29] CHUNG B,SHIN G W,NA J,et al.Multiplex quantitative foodborne pathogen detection using high resolution CE-SSCP coupled stuffer-free multiplex ligation-dependent probe amplification[J].Electrophoresis,2012,33(9/10):1477-1481.
[30] 陆长勇.食源性致病菌多重PCR 和基因芯片检测方法的建立[D].上海:上海交通大学,2009:17.
[31] 肖进文.猪肉及其制品中几种病原微生物芯片检测方法的建立及应用[J].安徽农业科学,2010,38(18):9478-9480.
[32] 祝儒刚,李拖平,宋立峰.应用基因芯片技术检测肉及肉制品中5 种致病菌[J].食品科学,2012,33(14):211-215.
[33] 商颖,许文涛,元延芳,等.通用引物多重PCR技术检测3种病原微生物[J].食品科学,2011,32(10):103-106.
[34] 刘忠梅,徐义刚,曲敏,等.3 种食源性致病菌Tem-PCR检测方法的建立[J].食品科学,2016,37(4):186-190.

备注/Memo

备注/Memo:
收稿日期:2019-02-14 基金项目:江苏省重点研发计划项目(BE2016648); 江苏省高校自然科学研究重大项目(16KJA210001) 作者简介:李同祥(1966-),男,教授,博士,主要从食品安全生物芯片检测技术研究.
更新日期/Last Update: 2019-05-30