电化学方法在李斯特氏菌毒力基因检测中的应用

doi:10.3724/SP.J.1005.2010.00512

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HEREDITAS ›› 2010, Vol. 32 ›› Issue (5): 512-516.doi: 10.3724/SP.J.1005.2010.00512

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Electrochemical detection of toxin gene in Listeria monocytogenes

WU Ling-Wei^{1, 2}, LIU Quan-Jun¹, WU Zhong-Wei¹, LU Zu-Hong¹

1. State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China;
2. Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, China

Received:2009-09-29 Revised:2009-11-30 Online:2010-05-20 Published:2010-05-15
Contact: LU Zu-Hong E-mail:zhlu@seu.edu.cn

Abstract

Abstract:

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

TUN Ling-Wei, LIU Quan-Dun, TUN Zhong-Wei, LIU Jie-Hong. Electrochemical detection of toxin gene in Listeria monocytogenes[J]. HEREDITAS, 2010, 32(5): 512-516.

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[1] Churchill RLT, Lee H, Hall JC. Detection of Listeria monocytogenes and the toxin listeriolysin O in food. J Microbiol Methods, 2006, 64(2): 141–170.

[3] Lunge VR, Miller BJ, Livak KJ, Batt CA. Factors affect-ing the performance of 5' nuclease

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Electrochemical detection of toxin gene in Listeria monocytogenes

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