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Diagnostic microarray for 14 water and foodborne pathogens using a flatbed scanner
Vidya Srinivasan1, Robert D Stedtfeld1, Dieter M Tourlousse1
1Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI 48824, United States.
Journal of Microbiological Methods
|April 26, 2017
Summary
Researchers developed a cost-effective microarray for simultaneous detection of 14 bacterial pathogens. This method uses gold labeling with silver enhancement (GLS) and multiplex polymerase chain reaction (PCR) for reliable, high-throughput screening.
Area of Science:
- Microbiology
- Biotechnology
- Molecular Diagnostics
Background:
- Simultaneous detection of multiple water and foodborne pathogens is crucial for public health.
- Limited resources in laboratories necessitate cost-effective and accessible diagnostic approaches.
- Existing methods may not offer the required throughput or affordability for widespread use.
Purpose of the Study:
- To develop and validate a high-density microarray for simultaneous screening of 14 bacterial pathogens.
- To establish a cost-effective and reliable method for pathogen detection suitable for resource-limited settings.
- To optimize probe design and data analysis for accurate presence/absence calls.
Main Methods:
- Designed 8887 50-mer probes targeting virulence and marker genes (VMGs) using an in-house database.
- Synthesized probes in quadruplicate on glass slides using in-situ synthesis technology.
- Generated target VMG amplicons via multiplex polymerase chain reaction (PCR), labeled with biotin, and hybridized to the microarray.
- Employed gold labeling with silver enhancement (GLS) protocol for signal generation.
- Quantified signals using a high-resolution flatbed scanner.
Main Results:
- Established reliable presence/absence call criteria: >4 probes/gene, signal-to-noise ratio (SNR) cutoff ≥2, and positive fraction (PF) >0.75.
- Achieved 100% correct calls with no false positives in blind sample testing.
- Demonstrated sensitivity comparable to standard PCR due to multiplex PCR amplification.
- Validated the cost-effectiveness and reliability of the GLS microarray approach.
Conclusions:
- The developed high-density microarray offers an inexpensive and reliable technique for high-throughput screening of multiple bacterial pathogens.
- This GLS-based approach is suitable for laboratories with limited resources, enhancing pathogen surveillance capabilities.
- The method provides accurate and sensitive detection, comparable to PCR, for simultaneous identification of various pathogens.
