Development of SYBR‐Green Real‐Time PCR and a Multichannel Electrochemical Immunosensor for Specific Detection of Salmonella enterica

Abstract

The objective of the present work was to develop and evaluate an SYBR Green real time polymerase chain reaction (PCR) method for the specific detection of Salmonella spp in broth cultures and meat samples experimentally contaminated. Also, a simple and rapid multichannel electrochemical immunosensor (MEI) for this pathogen is under study.

The PCR was carried out using primers ttr6 and ttr4 for the amplification of a highly conserved DNA region (ttr sequence) specific for all Salmonella serovars.

A boiling step, for the extraction of DNA, was combined with a real time PCR method based on the double‐stranded DNA (dsDNA) binding dye SYBR Green. The standard curve constructed using the mean threshold cycle and various concentrations of S. enteritidis (ranging fron 10² to 10⁸ CFU mL⁻¹) showed good linearity (R²=0.999) with the minimum level of detection of 10² CFU mL⁻¹. The experiments were conducted analyzing 30 Salmonella strains and 20 non‐Salmonella strains. All Salmonella serotypes tested were ttr‐positive and all other bacteria yielded no amplification products. The specificity of the reaction was confirmed by the melting temperature (T_m) of the amplicon obtained (T_m=80.1±0.1).

To verify the effectiveness of the assay, experiments were conducted on experimentally contaminated samples, which were also analyzed for comparison by the standard cultural method.

A multichannel electrochemical immunosensor for detection of Salmonella also was developed. It consists of a disposable screen‐printed sensor array, coupled with a multichannel pulse monitor, which was assembled as an immunosensor through the use of specific monoclonal (MAb) and polyclonal (PAb) antibodies in a sandwich format. The limit of detection was calculated to be 2×10⁶ UFC mL⁻¹ with a working range between 5×10⁶ to 5×10⁸ UFC mL⁻¹ and a total analysis time of about 3 h. This immunoelectrochemical system is economical, rapid, and easy to use but it is still under development in order to improve its analytical performance.

Keywords:

• PCR real‐time SYBR green
• Salmonella
• MEI