A sensitive assay for virus discovery in respiratory clinical samples.
In 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors...
| Main Authors: | , , , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2011-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3025933?pdf=render |
| _version_ | 1819108254507073536 |
|---|---|
| author | Michel de Vries Martin Deijs Marta Canuti Barbera D C van Schaik Nuno R Faria Martijn D B van de Garde Loes C M Jachimowski Maarten F Jebbink Marja Jakobs Angela C M Luyf Frank E J Coenjaerts Eric C J Claas Richard Molenkamp Sylvie M Koekkoek Christine Lammens Frank Leus Herman Goossens Margareta Ieven Frank Baas Lia van der Hoek |
| author_facet | Michel de Vries Martin Deijs Marta Canuti Barbera D C van Schaik Nuno R Faria Martijn D B van de Garde Loes C M Jachimowski Maarten F Jebbink Marja Jakobs Angela C M Luyf Frank E J Coenjaerts Eric C J Claas Richard Molenkamp Sylvie M Koekkoek Christine Lammens Frank Leus Herman Goossens Margareta Ieven Frank Baas Lia van der Hoek |
| author_sort | Michel de Vries |
| collection | DOAJ |
| description | In 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3'-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3-7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material. |
| first_indexed | 2024-12-22T03:07:01Z |
| format | Article |
| id | doaj.art-efe485a52c054e41ab1d9501e665d27e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-22T03:07:01Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-efe485a52c054e41ab1d9501e665d27e2022-12-21T18:41:02ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0161e1611810.1371/journal.pone.0016118A sensitive assay for virus discovery in respiratory clinical samples.Michel de VriesMartin DeijsMarta CanutiBarbera D C van SchaikNuno R FariaMartijn D B van de GardeLoes C M JachimowskiMaarten F JebbinkMarja JakobsAngela C M LuyfFrank E J CoenjaertsEric C J ClaasRichard MolenkampSylvie M KoekkoekChristine LammensFrank LeusHerman GoossensMargareta IevenFrank BaasLia van der HoekIn 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3'-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3-7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.http://europepmc.org/articles/PMC3025933?pdf=render |
| spellingShingle | Michel de Vries Martin Deijs Marta Canuti Barbera D C van Schaik Nuno R Faria Martijn D B van de Garde Loes C M Jachimowski Maarten F Jebbink Marja Jakobs Angela C M Luyf Frank E J Coenjaerts Eric C J Claas Richard Molenkamp Sylvie M Koekkoek Christine Lammens Frank Leus Herman Goossens Margareta Ieven Frank Baas Lia van der Hoek A sensitive assay for virus discovery in respiratory clinical samples. PLoS ONE |
| title | A sensitive assay for virus discovery in respiratory clinical samples. |
| title_full | A sensitive assay for virus discovery in respiratory clinical samples. |
| title_fullStr | A sensitive assay for virus discovery in respiratory clinical samples. |
| title_full_unstemmed | A sensitive assay for virus discovery in respiratory clinical samples. |
| title_short | A sensitive assay for virus discovery in respiratory clinical samples. |
| title_sort | sensitive assay for virus discovery in respiratory clinical samples |
| url | http://europepmc.org/articles/PMC3025933?pdf=render |
| work_keys_str_mv | AT micheldevries asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT martindeijs asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT martacanuti asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT barberadcvanschaik asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT nunorfaria asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT martijndbvandegarde asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT loescmjachimowski asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT maartenfjebbink asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT marjajakobs asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT angelacmluyf asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT frankejcoenjaerts asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT ericcjclaas asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT richardmolenkamp asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT sylviemkoekkoek asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT christinelammens asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT frankleus asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT hermangoossens asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT margaretaieven asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT frankbaas asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT liavanderhoek asensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT micheldevries sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT martindeijs sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT martacanuti sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT barberadcvanschaik sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT nunorfaria sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT martijndbvandegarde sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT loescmjachimowski sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT maartenfjebbink sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT marjajakobs sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT angelacmluyf sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT frankejcoenjaerts sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT ericcjclaas sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT richardmolenkamp sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT sylviemkoekkoek sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT christinelammens sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT frankleus sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT hermangoossens sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT margaretaieven sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT frankbaas sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples AT liavanderhoek sensitiveassayforvirusdiscoveryinrespiratoryclinicalsamples |