Detection of food spoilage and pathogenic bacteria based on ligation detection reaction coupled to flow-through hybridization on membranes

Abstract

Traditional culturing methods are still commonly applied for bacterial identification in the food control sector, despite being time and labor intensive. Microarray technologies represent an interesting alternative. However, they require higher costs and technical expertise, making them still inappropriate for microbial routine analysis. The present study describes the development of an efficient method for bacterial identification based on flow-through reverse dot-blot (FT-RDB) hybridization on membranes, coupled to the high specific ligation detection reaction (LDR). First, the methodology was optimized by testing different types of ligase enzymes, labeling, and membranes. Furthermore, specific oligonucleotide probes were designed based on the 16S rRNA gene, using the bioinformatic tool Oligonucleotide Retrieving for Molecular Applications (ORMA). Four probes were selected and synthesized, being specific for Aeromonas spp., Pseudomonas spp., Shewanella spp., and Morganella morganii, respectively. For the validation of the probes, 16 reference strains from type culture collections were tested by LDR and FT-RDB hybridization using universal arrays spotted onto membranes. In conclusion, the described methodology could be applied for the rapid, accurate, and cost-effective identification of bacterial species, exhibiting special relevance in food safety and quality.

Figure 1

Scheme of spotted zipcode-oligonucleotides on membranes for the optimization experiments. ⁶⁶Hybridization control; ⁶³ligation control.

Figure 2

Biodyne C membrane-macroarray images obtained for optimization experiments with different labeling: (a) biotin, (b) Cy3, and (c) Cy5 and zipcode-oligonucleotides spotted at different concentrations: (i) 1 μM, (ii) 0.1 μM, and (iii) 0.01 μM.

Figure 3

Nitrocellulose membrane-macroarray images obtained for optimization experiments with different labeling: (a) biotin, (b) Cy3, and (c) Cy5.

Figure 4

Biodyne C membrane-macroarray images obtained for optimization experiments with biotin labeling and different ligase enzymes: (a) pfu DNA ligase, (b) taq DNA ligase, and (c) ampligase.

Figure 5

Scheme of spotted zipcode-oligonucleotides on membranes. ^CColor control, ⁶⁶hybridization control, ⁶³ligation Control, ¹⁴ Pseudomonas spp., ¹⁷ Aeromonas spp., ²³ Shewanella spp., and ²⁹ Morganella morganii.

Figure 6

Biodyne C membrane-macroarray images obtained when testing the strains (a) Aeromonas hydrophila ATCC 7966, (b) Morganella morganii ATCC 8076, (c) Pseudomonas fluorescens ATCC 13525, (d) Shewanella putrefaciens ATCC 8071, (e) a mix of A. hydrophila ATCC 7966 and P. fluorescens ATCC 13525, and (f) a mix of all the four strains corresponding to the different species.