A DNA microarray for the versatile diagnosis of infectious diarrhea

Abstract

Several bacteria, viruses, and parasites cause diarrhea as coinfecting pathogens. We designed a DNA microarray comprising 60-bp probes spotted 194 times for the multiplex detection of 33 enteropathogenic bacteria and seven enteropathogenic viruses, and the archaeon Methanobrevibacter smithii was used as an internal positive control. Nine pathogen-free stool specimens were used as negative controls. One of these control specimens was further spiked with Salmonella enterica as a positive control. The microarray was then tested with 40 pathological stool specimens, comprising S. enterica (n = 30), Campylobacter jejuni (n = 4), pathogenic Escherichia coli (n = 2), and adenovirus (n = 4). M. smithii was detected in 47/49 (95.9%) specimens, no pathogen was detected in negative controls and S. enterica was identified in the S. enterica-spiked positive control. The overall specificity was 100% and the overall sensitivity was 97.5% because one S. enterica sample was missed by the microarray. The multiplexed detection of C. jejuni spiked into an adenovirus-positive stool sample gave positive results, with fluorescence values of 14.3 and 9.1, respectively. These data indicate that using the protocol developed in this article, the DNA array allows for the multiplexed detection of some enteropathogens in stool samples.

A Rapid LAMP-Based Method for Screening Poultry Samples for Campylobacter Without Enrichment

“…Rapid and robust testing systems are needed for implementation on-site to inform rapid action. Other methods for detection and identification of Campylobacter have been published in recent years ( Velusamy et al, 2010 ), including antibody-based detection ( Wadl et al, 2009 ), PCR ( Josefsen et al, 2004 ; Leblanc-Maridor et al, 2011 ; de Boer et al, 2015 ), DNA microarrays ( Quinones et al, 2007 ; Donatin et al, 2013 ), and loop-mediated isothermal DNA amplification (LAMP) ( Yamazaki et al, 2009 ; Dong et al, 2014 ; Sabike et al, 2016 ). Nevertheless, most procedures are still not fully adequate to be deployed on-site, as they involve specialized skills or facilities to perform certain steps such as DNA extraction or enrichment culture.…”

Optimized microbial DNA extraction from diarrheic stools

“…The protocol here reported, combining lyophilization and a semi-automated DNA extraction, could be used for the routine detection of enteropathogen DNA in diarrheal stool specimens and the molecular diagnosis of infectious diarrhea [24]. …”

DNA Microarrays for Pathogen Detection

“…Several research groups developed DNA microarray tests for the detection of enteropathogenic bacteria directly from human diarrheal stool samples as an alternative to traditional culture-based methods. Donatin et al [25] developed a DNA microarray for the detection of 33 enteropathogenic bacteria and 7 enteropathogenic viruses. Genomic DNA extracted from stool samples was fluorescence-labeled and hybridized on the microarray without prior amplification, but with a rather long hybridization time of 40 h. The microarray was tested with 40 pathological stool specimens, showing an overall specificity of 100% and an overall sensitivity of 97.5%.…”