|Suo, Biao - Shanghai Jiaotong University|
|Tu, Shu I|
|Shi, Xianming - Shanghai Jiaotong University|
Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/6/2009
Publication Date: 10/13/2009
Citation: Suo, B., He, Y., Paoli, G., Gehring, A.G., Tu, S., Shi, X. 2009. Development of an oligonucleotide-based microarray to detect multiple foodborne pathogens. Molecular and Cellular Probes. 24:77-86. Interpretive Summary: Foodborne illness remains a public health burden in the United States. To prevent food-related illnesses and improve food safety, effectively monitoring microbial contaminations in food environments and supplies is a key. In the present study, we have used advanced molecular techniques to develop a powerful and cost-effective assay for multiple foodborne pathogen detection. The assay was able to simultaneously screen up to 12 independent samples and identify four major foodborne pathogens including Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes and Campylobacter jejuni. In addition to pathogen detection, the assay also provided important virulence information related to the pathogens at the same analysis. Therefore, the multiplex assay developed in this study may be suitable for large-scale inspections of multiple pathogen occurrences.
Technical Abstract: Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes and Campylobacter jejuni are considered important human pathogens causing the most food-related human illnesses worldwide. Current methods for pathogen detection have limitations in effectively identifying multiple foodborne pathogens. In this study, a pathogen detection microarray was developed using various 70-mer oligonucleotides specifically targeting the above pathogens. To reduce the cost of detection, each microarray chip was designed and fabricated to accommodate 12 identical arrays which could be used for screening up to 12 different samples. To achieve high detection sensitivity and specificity, different DNA pre-amplification strategies including target specific multiplex PCR and whole genome random amplifications were compared by hybridizing amplicons to the detection microarray. The target-specific amplification resulted in higher specificity in microarray detection compared to the random amplifications. The multiplex PCR was able to simultaneously amplify 14 target nucleotide sequences from the above pathogens in a single reaction tube. This significantly improved the efficiency of the assay from singleplex PCR. Combined with multiplex PCR amplification of target sequences, the microarray unambiguously distinguished all 4 pathogens on the same array with a detection sensitivity of 1x10-4 ng (approximately 20 copies) of each genomic DNA. This method not only greatly improved the efficacy for simultaneous detection of multiple foodborne pathogens but also provided important genotypic information related to pathogen virulence on the same array.