Journal
JOURNAL OF MICROBIOLOGICAL METHODS
Volume 139, Issue -, Pages 15-21Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.mimet.2017.04.009
Keywords
Waterborne pathogens; Gold labeling and silver enhancement; Virulence and marker genes; Low cost microarray
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Funding
- National Institute of Health [5 R01 RR018625-03]
- Michigan Economic Development Corporation [GR-476 PO 085P3200517]
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Parallel detection approaches are of interest to many researchers interested in identifying multiple water and foodborne pathogens simultaneously. Availability and cost-effectiveness are two key factors determining the usefulness of such approaches for laboratories with limited resources. In this study, we developed and validated a high-density microarray for simultaneous screening of 14 bacterial pathogens using an approach that employs gold labeling with silver enhancement (GLS) protocol. In total, 8887 probes (50-mer) were designed using an inhouse database of virulence and marker genes (VMGs), and synthesized in quadruplicate on glass slides using an in -situ synthesis technology. Target VMG amplicons were obtained using multiplex polymerase chain reaction (PCR), labeled with biotin, and hybridized to the microarray. The signals generated after gold deposition and silver enhancement, were quantified using a flatbed scanner having 2-mu m resolution. Data analysis indicated that reliable presence/absence calls could be made, if: i) over four probes were used per gene, ii) the signal-to-noise ratio (SNR) cutoff was greater than or equal to two, and iii) the positive fraction (PF), Le., number of probes with SNR 2 for a given VMG was greater than 0.75. Hybridization of the array with blind samples resulted in 100% correct calls, and no false positive. Because amplicons were obtained by multiplex PCR, sensitivity of this method is similar to PCR. This assay is an inexpensive and reliable technique for high throughput screening of multiple pathogens.
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