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Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction
Published on: November 1, 2018
Accurate strand-specific quantification of viral RNA
Nicole E Plaskon1, Zach N Adelman, Kevin M Myles
1Fralin Life Science Institute, Department of Entomology, Virginia Tech, Blacksburg, Virginia, United States of America.
Plos One
|October 23, 2009
Summary
Accurate detection of viral RNA strands is crucial for studying RNA viruses. Standard strand-specific quantitative real-time PCR (ssqPCR) assays struggle with accuracy, but tag-specific primer assays improve quantification of viral RNA strands.
Area of Science:
- Virology
- Molecular Biology
- Biotechnology
Background:
- Accurate detection of viral RNA strands is essential for understanding RNA virus replication.
- Strand-specific quantitative real-time PCR (ssqPCR) is a common method, but its accuracy is debated.
- Existing ssqPCR assays may not reliably quantify viral RNA strands due to primer-related issues.
Purpose of the Study:
- To evaluate the accuracy of different ssqPCR assay designs for quantifying viral RNA strands.
- To compare the performance of standard and tag-specific primer ssqPCR assays.
- To develop and validate reliable ssqPCR assays for o'nyong-nyong virus (ONNV) and chikungunya virus (CHIKV).
Main Methods:
- Development of multiple ssqPCR assays for ONNV based on prevalent literature designs.
- Comparison of assay parameters, including standard virus-specific primers versus tag-specific primers.
- Evaluation of SYBR Green and DNA hydrolysis probes for detection sensitivity.
Main Results:
- Standard ssqPCR assays failed to distinguish between true and false priming, hindering accurate quantification.
- Inaccurate quantification of ONNV (-) strand RNA was observed with standard assays when (+) strand cDNA was abundant.
- Tag-specific primer assays demonstrated accurate quantification of ONNV (-) strand RNA, overcoming limitations of standard assays.
Conclusions:
- Standard ssqPCR assays may not accurately quantify anti-genomic viral RNA strands during infection cycles.
- Tag-specific primer assays offer a more reliable method for accurate quantification of viral RNA strands.
- Validated ssqPCR assays for ONNV and CHIKV can aid in studying these significant RNA viruses.
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