Detection of hlyA Gene of Listeria Monocytogenes with Electrochemical DNA Biosensor

Listeria monocytogenes (LM) is a bacterial foodborne pathogen responsible for listeriosis, an illness characterized by encephalitis, septicaemia , and meningitis. One of the best ways to detect and confrim the pathogen is through the detection of one of the virulence factors, listeriolysin O (LLO) produced by the microorganism. LLO is absolutely required for virulence by L. monocytogenes, and is found only in virulent strains of the species. This paper focuses on the electrical detection methods used to detect the LLO toxin gene in food products or organism. A new and simple electrochemical approach for hybridisation detection without the labelling the target DNA is described. The coupling of DNA electrochemical sensors have specialities of quick detection and cheapness and have the potential of the quantitative evaluation of the hlyA gene of Listeria monocytogenes. The electrochemical sensor was obtained by immobilizing single-stranded oligonucleotides onto gold electrodes through mercaptan of the DNA bases using N- hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl-N´-ethyl carbodiimidehydrochloride (EDC) as activation. The hybridization reaction that occurred on the electrode surface was evidenced by cyclic voltammetry (CV) analysis - using as [Co(phen)₃](C1O₄)₃ indicator. The covalently immobilized single-stranded DNA could selectively hybridize with its complementary DNA in solution to form double-stranded DNA on the gold surface. A significant increase of the peak current of cyclic voltammetry (CV) upon the hybridization of immobilized ssDNA with PCR amplification products in the solution was observed. This peak current change was used to monitor the amount of PCR amplification products. Factors such as DNA target concentration and hybridization conditions determining the sensitivity of the electrochemical assay were investigated.