电化学方法在李斯特氏菌毒力基因检测中的应用
收稿日期: 2009-09-29
修回日期: 2009-11-30
网络出版日期: 2010-05-15
基金资助
国家自然科学基金项目(编号：60121101, 60671019)和江西省自然科学基金项目(编号：2008GZN0036)资助
Electrochemical detection of toxin gene in Listeria monocytogenes
Received date: 2009-09-29
Revised date: 2009-11-30
Online published: 2010-05-15
单核李斯特氏菌(Listeria monocytogenes, LM)是食源性李斯特氏病的病源菌, 该病可引起败血病、脑膜炎、流产等。李斯特氏菌的毒力因子listeriolysin O (LLO)是引发李斯特氏病的主要原因。文章使用一种特殊的电化学方法从样品中检测编码LLO的hlyA基因。该方法以化合物Nhydroxysulfosuccinimide (NHS) 和 N-(3-dimethylamion) propyl- N'-ethyl carbodiimidehydrochloride (EDC) 作为激活剂, 使单链DNA探针结合到金电极表面组成工作电极, 以[Co(phen)3](ClO4)3 作为指示剂来检测循环伏安曲线(Cyclic voltammetry , CV), 通过CV峰值的变化来估算hlyA基因的含量, 从而确定LM的污染情况。这种新颖的电化学方法用于免标记的目标DNA的杂交检测, 具有快速和方便的特点。
吴凌伟，刘全俊，吴中伟，陆祖宏 . 电化学方法在李斯特氏菌毒力基因检测中的应用[J]. 遗传, 2010 , 32(5) : 512 -516 . DOI: 10.3724/SP.J.1005.2010.00512
Listeria monocytogenes (LM) is a food-borne pathogen inducing listeriosis, an illness characterized by encephalitis, septicaemia, and meningitis. Listeriolysin O (LLO) is absolutely required for virulence by L. monocytogenes, and is found only in virulent strains of the species. One of the best ways to detect and confirm the pathogen is detection of one of the virulence factors, LLO, produced by the microorganism. This paper focused on the electrical method used to detect the LLO toxin gene in food products and organism without labeling the target DNA. The electrochemical sensor was obtained by immobilizing single-stranded oligonucleotides onto the gold electrode with the mercaptan activated by N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl-N'-ethyl carbodiimidehydrochloride (EDC). The hy-bridization reaction that occurred on the electrode surface was evidenced by Cyclic Voltammetry (CV) analysis using [Co(phen)3](ClO4)3 as an indicator. The covalently immobilized single-stranded DNA could selectively hybridize to its complementary DNA in solution to form double-stranded DNA on the gold surface. A significant increase of the peak cur-rent of Cyclic Voltammetry (CV) upon hybridization of immobilized ssDNA with PCR amplification products in the solu-tion was observed. This peak current change was used to monitor the amount of PCR amplification products. Factors deter-mining the sensitivity of the electrochemical assay, such as DNA target concentration and hybridization conditions, were investigated. The coupling of DNA to the electrochemical sensors has the potential of the quantitative evaluation of gene.
Key words： Listeria monocytogenes; electrochemical assay; DNA detection; HlyA gene
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[2] Hudson JA, Lake RJ, Savill MG, Scholes P, McCormick RE. Rapid detection of Listeria monocytogenes in ham samples using immunomagnetic separation followed by polymerase chain reaction. J Appl Microbiol, 2001, 90(4): 614–621.
[3] Lunge VR, Miller BJ, Livak KJ, Batt CA. Factors affect-ing the performance of 5' nuclease
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