Development of multiplex oligonucleotide ligation-PCR-universal DNA microarrays for detection of foodborne pathogens
Wang, X.; Ying, S.; Han, R.; Yuan, J.
Wei Sheng Yan Jiu 46(2): 225-231
2017
ISSN/ISBN: 1000-8020 PMID: 29903098 Document Number: 693177
To develop a multiplex oligonucleotide ligation-polymerase chain reaction( MOL-PCR) based universal microarray assay for multiplexed detection of foodborne pathogens. 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. 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. The assay provides a novel platform for rapid, accurate, sensitive and high-throughput detection of pathogenic bacteria of foodborne diseases.