Accurate strand-specific quantification of viral RNA.
The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitativ...
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Public Library of Science (PLoS)
2009-10-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0007468&type=printable |
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| _version_ | 1849695120317743104 |
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| author | Nicole E Plaskon Zach N Adelman Kevin M Myles |
| author_facet | Nicole E Plaskon Zach N Adelman Kevin M Myles |
| author_sort | Nicole E Plaskon |
| collection | DOAJ |
| description | The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o'nyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (-) strand RNA with this assay when higher levels of cDNA generated from the (+) strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (-) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR(R) Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region. |
| format | Article |
| id | doaj-art-686c5d3dbe244d23bc9f4774b2c1f01b |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2009-10-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-686c5d3dbe244d23bc9f4774b2c1f01b2025-08-20T03:19:52ZengPublic Library of Science (PLoS)PLoS ONE1932-62032009-10-01410e746810.1371/journal.pone.0007468Accurate strand-specific quantification of viral RNA.Nicole E PlaskonZach N AdelmanKevin M MylesThe presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o'nyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (-) strand RNA with this assay when higher levels of cDNA generated from the (+) strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (-) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR(R) Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0007468&type=printable |
| spellingShingle | Nicole E Plaskon Zach N Adelman Kevin M Myles Accurate strand-specific quantification of viral RNA. PLoS ONE |
| title | Accurate strand-specific quantification of viral RNA. |
| title_full | Accurate strand-specific quantification of viral RNA. |
| title_fullStr | Accurate strand-specific quantification of viral RNA. |
| title_full_unstemmed | Accurate strand-specific quantification of viral RNA. |
| title_short | Accurate strand-specific quantification of viral RNA. |
| title_sort | accurate strand specific quantification of viral rna |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0007468&type=printable |
| work_keys_str_mv | AT nicoleeplaskon accuratestrandspecificquantificationofviralrna AT zachnadelman accuratestrandspecificquantificationofviralrna AT kevinmmyles accuratestrandspecificquantificationofviralrna |