Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization.
Human norovirus (NoV) is the leading cause of acute viral gastroenteritis and a major source of foodborne illness. Detection of NoV in food and environmental samples is typically performed using molecular techniques, including real-time reverse transcription polymerase chain reaction (RT-PCR) and le...
Saved in:
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2021-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0248581&type=printable |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850046436841881600 |
|---|---|
| author | Clyde S Manuel Cassandra Suther Matthew D Moore Lee-Ann Jaykus |
| author_facet | Clyde S Manuel Cassandra Suther Matthew D Moore Lee-Ann Jaykus |
| author_sort | Clyde S Manuel |
| collection | DOAJ |
| description | Human norovirus (NoV) is the leading cause of acute viral gastroenteritis and a major source of foodborne illness. Detection of NoV in food and environmental samples is typically performed using molecular techniques, including real-time reverse transcription polymerase chain reaction (RT-PCR) and less frequently, nested real-time PCR. In this study, we conducted a controlled comparison of two published NoV detection assays: a broadly reactive one-step real-time RT-PCR and a two-step nested real-time PCR assay. A 20% human fecal suspension containing a genogroup II human NoV was serially diluted, genome extracted, and subjected to amplification using the two assays compared via PCR Units. Additional amplicon confirmation was performed by dot blot hybridization using digoxigenin (DIG)-labeled oligonucleotide probes. Both assays displayed similar amplification standard curves/amplification efficiencies; however, the nested assay consistently detected one log10 lower virus. Dot blot hybridization improved the detection limit of the nested real-time PCR by one log10 NoV genome copies but impaired the detection limit of the one-step real-time RT-PCR by one log10 NoV genome copies. These results illustrate the complexities in designing and interpreting molecular techniques having a sufficient detection limit to detect low levels of viruses that might be anticipated in contaminated food and environmental samples. |
| format | Article |
| id | doaj-art-e70038453cd241088b58b429be7aba39 |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2021-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-e70038453cd241088b58b429be7aba392025-08-20T02:54:28ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-01164e024858110.1371/journal.pone.0248581Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization.Clyde S ManuelCassandra SutherMatthew D MooreLee-Ann JaykusHuman norovirus (NoV) is the leading cause of acute viral gastroenteritis and a major source of foodborne illness. Detection of NoV in food and environmental samples is typically performed using molecular techniques, including real-time reverse transcription polymerase chain reaction (RT-PCR) and less frequently, nested real-time PCR. In this study, we conducted a controlled comparison of two published NoV detection assays: a broadly reactive one-step real-time RT-PCR and a two-step nested real-time PCR assay. A 20% human fecal suspension containing a genogroup II human NoV was serially diluted, genome extracted, and subjected to amplification using the two assays compared via PCR Units. Additional amplicon confirmation was performed by dot blot hybridization using digoxigenin (DIG)-labeled oligonucleotide probes. Both assays displayed similar amplification standard curves/amplification efficiencies; however, the nested assay consistently detected one log10 lower virus. Dot blot hybridization improved the detection limit of the nested real-time PCR by one log10 NoV genome copies but impaired the detection limit of the one-step real-time RT-PCR by one log10 NoV genome copies. These results illustrate the complexities in designing and interpreting molecular techniques having a sufficient detection limit to detect low levels of viruses that might be anticipated in contaminated food and environmental samples.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0248581&type=printable |
| spellingShingle | Clyde S Manuel Cassandra Suther Matthew D Moore Lee-Ann Jaykus Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization. PLoS ONE |
| title | Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization. |
| title_full | Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization. |
| title_fullStr | Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization. |
| title_full_unstemmed | Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization. |
| title_short | Comparison of a one-step real-time RT-PCR and a nested real-time RT-PCR for a genogroup II norovirus reveals differences in sensitivity depending upon assay design and visualization. |
| title_sort | comparison of a one step real time rt pcr and a nested real time rt pcr for a genogroup ii norovirus reveals differences in sensitivity depending upon assay design and visualization |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0248581&type=printable |
| work_keys_str_mv | AT clydesmanuel comparisonofaonesteprealtimertpcrandanestedrealtimertpcrforagenogroupiinorovirusrevealsdifferencesinsensitivitydependinguponassaydesignandvisualization AT cassandrasuther comparisonofaonesteprealtimertpcrandanestedrealtimertpcrforagenogroupiinorovirusrevealsdifferencesinsensitivitydependinguponassaydesignandvisualization AT matthewdmoore comparisonofaonesteprealtimertpcrandanestedrealtimertpcrforagenogroupiinorovirusrevealsdifferencesinsensitivitydependinguponassaydesignandvisualization AT leeannjaykus comparisonofaonesteprealtimertpcrandanestedrealtimertpcrforagenogroupiinorovirusrevealsdifferencesinsensitivitydependinguponassaydesignandvisualization |