RESUMO
Bacterial foodborne diseases remain major threats to food safety and public health, especially in developing countries. In this study a novel assay, combining gold nanoparticle (GNP)-based multiplex oligonucleotide ligation-PCR and universal oligonucleotide microarray technology, was developed for inexpensive, specific, sensitive, and multiplex detection of eight common foodborne pathogens, including Shigella spp., Campylobacter jejuni, Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus, and Vibrio parahaemolyticus. The target fragments of the eight pathogens were enriched by multiplex PCR and subjected to multiplex ligase detection reaction. Ligation products were enriched and labeled with GNPs by universal asymmetric PCR, using excess GNP-conjugated primers. The labeled single-stranded amplicons containing complementary tag sequences were captured by the corresponding tag sequences immobilized on microarrays, followed by silver staining for signal enhancement. Black images of microarray spots were visualized by naked eyes or scanned on a simple flatbed scanner, and quantified. The results indicated that this assay could unambiguously discriminate all eight pathogens in single and multiple infections, with detection sensitivity of 3.3-85CFU/mL for pure cultures. Microarray results of ninety-five artificially contaminated and retail food samples were consistent with traditional culture, biochemical and real-time PCR findings. Therefore, the novel assay has the potential to be used for routine detection due to rapidity, low cost, and high specificity and sensitivity.
Assuntos
Microbiologia de Alimentos/métodos , Inocuidade dos Alimentos/métodos , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Bacillus cereus/genética , Bacillus cereus/isolamento & purificação , Campylobacter jejuni/genética , Campylobacter jejuni/isolamento & purificação , Primers do DNA/genética , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Microbiologia de Alimentos/instrumentação , Ouro , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Nanopartículas Metálicas , Reação em Cadeia da Polimerase Multiplex/métodos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Salmonella enterica/genética , Salmonella enterica/isolamento & purificação , Shigella/genética , Shigella/isolamento & purificação , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/isolamento & purificaçãoRESUMO
OBJECTIVE: To develop a multiplex oligonucleotide ligation-polymerase chain reaction( MOL-PCR) based universal microarray assay for multiplexed detection of foodborne pathogens. METHODS: Eight common foodborne pathogens causing bacterial food poisoning were selected as detection models. An upstream and downstream adjacent detection probes were designed within specific primer pair for each of eight pathogens. Target fragments of the eight pathogens were enriched by multiplex PCR and used as ligation templates. Abundant fluorescently labeled single-stranded amplicons containing anti-tag sequences were gained by multiplex ligase detection reaction and asymmetric PCR labeling with universal primers. The products could be detected by hybridization with corresponding tag sequences immobilized on DNA microarrays. RESULTS: The results indicated that the assay could specifically identify all eight pathogens in single and multiple infections. The detection limits were( 1. 1- 8. 5) × 10~2 CFU /mL of pure bacterial cultures. The microarray results for 96 food poisoning and clinical diarrheal samples were consistent with that of traditional culture, biochemical identification and real-time PCR. CONCLUSION: The assay provides a novel platform for rapid, accurate, sensitive and high-throughput detection of pathogenic bacteria of foodborne diseases.
