Detection of eight foodborne bacterial pathogens by oligonucleotide array hybridization

Background: Simultaneous and rapid detection of multiple foodborne bacterial pathogens is important for the prevention of foodborne illnesses. Objective: The aim of this study was to evaluate the use of 16S rDNA and 23S rDNA sequences as targets for simultaneous detection of eight foodborne bacterial pathogens. Methods: Nineteen bacterial oligonucleotide probes were synthesized and applied to nylon membranes. Digoxygenin labeled 16S rDNA and 23S rDNA from bacteria were amplified by PCR using universal primers, and the amplicons were hybridized to the membrane array. Hybridization signals were visualized by NBT/BCIP color development. Results: The eight intestinal bacterial pathogens including Salmonella enterica, Escherichia coli, Bacillus cereus, Vibrio cholerae, Shigella dysenteriae, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis were appropriately detected in a panel of oligonucleotide array hybridization. The experimental results showed that the method could discriminate the bacterial pathogens successfully. The sensitivity of oligonucleotide array was 103 CFU/ml. Conclusion: This study showed that 16S rDNA and 23S rDNA genes had sufficient sequence diversity for species identification and were useful for monitoring the populations of foodborne pathogenic bacteria. Furthermore, results obtained in this study revealed that oligonucleotide array hybridization had a powerful capability to detect and identify the bacterial pathogens simultaneously

Detection of eight foodborne bacterial pathogens by oligonucleotide array hybridization

Background: Simultaneous and rapid detection of multiple foodborne bacterial pathogens is important for the prevention of foodborne illnesses. Objective: The aim of this study was to evaluate the use of 16S rDNA and 23S rDNA sequences as targets for simultaneous detection of eight foodborne bacterial pathogens. Methods: Nineteen bacterial oligonucleotide probes were synthesized and applied to nylon membranes. Digoxygenin labeled 16S rDNA and 23S rDNA from bacteria were amplified by PCR using universal primers, and the amplicons were hybridized to the membrane array. Hybridization signals were visualized by NBT/BCIP color development. Results: The eight intestinal bacterial pathogens including Salmonella enterica, Escherichia coli, Bacillus cereus, Vibrio cholerae, Shigella dysenteriae, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis were appropriately detected in a panel of oligonucleotide array hybridization. The experimental results showed that the method could discriminate the bacterial pathogens successfully. The sensitivity of oligonucleotide array was 103 CFU/ml. Conclusion: This study showed that 16S rDNA and 23S rDNA genes had sufficient sequence diversity for species identification and were useful for monitoring the populations of foodborne pathogenic bacteria. Furthermore, results obtained in this study revealed that oligonucleotide array hybridization had a powerful capability to detect and identify the bacterial pathogens simultaneously

Detection of Escherichia coli, Salmonella enterica and shigella Dysenteriae by analysis of 23s ribosomal DNA gene

Background: Ribosomal DNA (rDNA) genes contain signature structures which are unique for groups of organisms. Considering their great number in cells and their protected areas, they render ideal targets for specific nucleic acid probes. The present study aimed to investigate the capability of some specific regions of 23S rDNA gene as a DNA target for differentiation and screening of Escherichia coli, Salmonella enterica, and Shigella dysenteriae. Methods: Bacterial reference strains used in this study were E. coli, S. enterica and Sh. dysenteriae that were provided by the centers for microbial culture collection (CMCC) at Pasture Institute of Iran. DNA extraction was performed by boiling method. Alignment of the 23S rDNA sequences of bacterial species was performed by using AlignX (a component of Vector NTI Advance 11.0) and areas displaying sequence divergence among species were used for designing universal primers and individual bacteria specific probe. Findings: The universal polymerase chain reaction (PCR) products of each bacterial species showed bands of approximately 880 bp to be being equivalent to the fragment size of 23S rDNA gene. Different size bands of 23S rDNA probes were produced and included 228 bp for E. coli, 444 bp for S. enterica, and 776 bp for Sh. dysenteriae. Conclusion: Comparative sequence analysis of variable and specific regions of 23S rDNA genes among the studied bacterial species showed that we were able to amplify specific target among universal region for the detection of many enteric pathogenic bacteria