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High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
Published on: August 3, 2011
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Optimizing a custom tiling microarray for low input detection and identification of unamplified virus targets.
Christine Yu1, Samantha Q Wales1, Mark K Mammel1
1Division of Molecular Biology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708, USA.
Journal of Virological Methods
|April 2, 2016
Summary
Sensitive DNA microarrays can detect low levels of foodborne viruses like hepatitis A, norovirus, and coxsackievirus without amplification. This method offers a promising approach for identifying viral contamination in food samples.
Area of Science:
- Food safety
- Virology
- Molecular diagnostics
Background:
- Foodborne viruses are significant pathogens, often present at low levels, necessitating sensitive detection methods.
- Traditional culture methods are inefficient for many viruses, and multi-species contamination is common.
- Non-amplification-based detection is crucial for accurate foodborne virus identification.
Purpose of the Study:
- To evaluate a custom DNA microarray (FDA_EVIR) for sensitive detection of low-input foodborne viruses.
- To assess the array's capability in identifying hepatitis A virus, norovirus, and coxsackievirus, individually and combined.
- To optimize sample processing for unamplified, low-level viral targets.
Main Methods:
- Utilized a custom DNA microarray (FDA_EVIR) for amplification-free virus detection.
- Modified sample processing with carrier cDNA, RNase, and DNase I fragmentation for low-input targets.
- Employed alternative data analysis for genotype distinction and minority species identification.
Main Results:
- Achieved amplification-free detection and identification of foodborne viruses at 250-500 copies of viral RNA.
- Successfully distinguished virus genotypes, especially at low target levels.
- Identified minority viral species within mixed infections using single probe analysis.
Conclusions:
- The custom DNA microarray (FDA_EVIR) is a promising platform for detecting foodborne viruses at low levels.
- The amplification-free approach enhances sensitivity and accuracy in complex food matrices.
- This method supports robust identification of viral pathogens in food and environmental samples.
