纳米金比色芯片检测和鉴定病原微生物-东南大学学报医学版

纳米金比色芯片检测和鉴定病原微生物

单位：1.东南大学生物医学工程系分子与生物分子电子学教育部重点实验室,江苏,南京,210096;江苏省疾病预防控制中心,江苏,南京,210009； 2.东南大学生物医学工程系分子与生物分子电子学教育部重点实验室,江苏,南京,210096； 3.江苏省疾病预防控制中心,江苏,南京,210009

关键词：比色芯片链霉亲和素-纳米金聚合酶链反应/方法大肠杆菌O158/分离和提纯

分类号：R319, R378.2, R446.9

出版年·卷·期（页码）：2004·23·第一期（18-22）

摘要：

目的:建立一种简单、快速的比色芯片判断PCR产物存在与否的方法.方法:在多重PCR过程中掺入生物素化的dUTP (biotin-16-dUTP )制备生物素化的靶序列,与相应的生物芯片杂交后,用链霉亲和素-纳米金结合银染的方法鉴定4种不同大肠杆菌血清型.同时,为了与链霉亲和素-藻红蛋白染色方法进行比较,用这两种方法检测了大肠杆菌血清型O157:H7的rfbE扩增产物.结果:纳米金比色芯片检测结果与琼脂糖凝胶电泳的结果一致,芯片杂交的纳米金标记与荧光标记检测的结果相当.结论:纳米金比色芯片能够对4种不同的大肠杆菌血清型进行准确鉴定,而且不需复杂的仪器,适合现场检测监控环境中的微生物.

Objective To establish a simple and rapid colorimetric silver staining method for detecting nucleic acid hybridization on DNA microarray.Methods The biotinylated fragments of target DNA were prepared by incorporating the biotin-labeled nucleotide during multiplex PCR amplification,and were used to hybridize with the array without further modification or purification.After the hybridization,the PCR products were directly detected with a streptavidin-phycoerythrin conjugate or detected with a streptavidin labeled with nanogold particles and the signal were amplified by silver precipitation.We also compared the ability of the two methods for detecting a 149 bp rfbE PCR product of E.coli O157H7.Results and Conclusion These results indicated that this colorimetric assays can identify accurately four various serotype of the E.coli,and is suited for field monitoring the pathogens in environment without complicated instrument.

参考文献：

[1] Rajeevan M S, DIMULESCU I M, UNGER E R. Chemiluminescent analysis of gene expression on high density filter arrays. 1999(3)
[2] Chen J W, WU R, Yang P C. Profiling expression patterns and isolating differentially expressed genes by cDNA microarray system with colorimetry detection. 1998(3). doi:10.1006/geno.1998.5354
[3] Alexandre I, HAMELS S, DUFOUR S. Colorimetric silver detection of DNA microarrays. 2001(1). doi:10.1006/abio.2001.5176
[4] LEVIT-BINNUN N, LINDNER A B, ZIK O. Quantitative detection of protein arrays. 2003(6). doi:10.1021/ac0261350
[5] Wang J, SONG J Y, ZHOU F M. Silver-enhanced imaging of DNA hybridization at DNA microarrays with scanning electrochemical microscopy. 2002(17). doi:10.1021/la011822s
[6] Riley L W, REMIS R S, HELGERSON S D. Hemorrhagic colitis associated with a rare Escherichia coli serotype, 1983
[7] BESSER R E, GRIFFIN P M, SLUTSKER L. Escherichia coli O157:H7 gastroenteritis and the hemolytic uremic syndrome:an emerging infectious disease. 1999(). doi:10.1146/annurev.med.50.1.355
[8] Fortin N Y, MULCHANDANI A, Chen W. Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli O157:H7. 2001(2). doi:10.1006/abio.2000.4935
[9] Chizhikov V, RASOOLY A, CHUMAKOV K. Microarray analysis of microbial virulence factors. 2001(7)
[10] Paton A W, PATON J C. Detection and characterization of shiga toxigenic Escherichia coili by using multiplex PCR assays for stx Ⅰ,stx Ⅱ,eaeA,enterohemorrhagic E.coli hlyA,rfbo111,rfbo157. 1998(2)

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