Efficiency of Automated Viral RNA Purification for Pediatric Studies of Dengue and Zika in Hyperendemic Areas
The isolation of nucleic acids is a critical and limiting step for molecular assays, which prompted the arrival in Colombia of automated purification instruments during the SARS-CoV-2 pandemic. The local application of this technology in the study of tropical diseases, such as dengue and zika, is be...
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2023
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ftdoajarticles:oai:doaj.org/article:597622845a2c461dbbaa41627f5978b8 2023-11-12T04:14:02+01:00 Efficiency of Automated Viral RNA Purification for Pediatric Studies of Dengue and Zika in Hyperendemic Areas Sandra L. Delgado Piedad M. Perilla Doris M. Salgado María Clemencia Rojas Carlos F. Narváez 2023-01-01T00:00:00Z https://doi.org/10.1155/2023/1576481 https://doaj.org/article/597622845a2c461dbbaa41627f5978b8 EN eng Hindawi Limited http://dx.doi.org/10.1155/2023/1576481 https://doaj.org/toc/1687-9694 1687-9694 doi:10.1155/2023/1576481 https://doaj.org/article/597622845a2c461dbbaa41627f5978b8 Journal of Tropical Medicine, Vol 2023 (2023) Arctic medicine. Tropical medicine RC955-962 article 2023 ftdoajarticles https://doi.org/10.1155/2023/1576481 2023-10-15T00:40:01Z The isolation of nucleic acids is a critical and limiting step for molecular assays, which prompted the arrival in Colombia of automated purification instruments during the SARS-CoV-2 pandemic. The local application of this technology in the study of tropical diseases, such as dengue and zika, is beginning to be tested. We evaluated the efficiency of the automated extraction of viral RNA for studies of pediatric dengue and zika. Clinical samples of children with dengue that were well characterized through RNA isolation by silica columns and serotype-specific nested RT-PCR (DENV-1 n = 7, DENV-2 n = 5, and negatives n = 8) in addition to 40 pediatric plasma samples spiked with ZIKV (strain PRVA BC59) and 209 from negative pre-epidemic children were analyzed. RNA from patients was extracted by two automated standard and high-throughput protocols on the KingFisher™ Flex instrument. The isolated RNA was evaluated for concentration and purity by spectrophotometry, for structural and functional integrity by electrophoresis and expression of the RNase P gene, and usefulness in serotype-specific DENV detection by conventional and real-time RT-PCR. For the evaluation of ZIKV RNA, the commercial TaqMan Triplex® assay was used, along with a well-tested in-house RT-qPCR assay. The concentration of RNA (5.2 vs. 7.5 ng/μL, P=0.03) and the number of integral bands (9 vs. 11) were higher with the high-throughput protocol. However, the number of specimens serotyped for DENV by RT-qPCR was comparable for both protocols. The cycle thresholds of the TaqMan Triplex® commercial kit and the in-house assay for the detection of plasma ZIKV RNA isolated with the standard protocol showed a strong association (r = 0.93, P<0.0001) and a Cohen Kappa index of 0.98 when all 249 samples were analyzed. These preliminary results suggest that automated instruments could be used in studies of cocirculating flaviviruses that have represented a public health problem in recent decades in Colombia. They boast advantages such as efficiency, precision, ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Journal of Tropical Medicine 2023 1 11 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Sandra L. Delgado Piedad M. Perilla Doris M. Salgado María Clemencia Rojas Carlos F. Narváez Efficiency of Automated Viral RNA Purification for Pediatric Studies of Dengue and Zika in Hyperendemic Areas |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 |
description |
The isolation of nucleic acids is a critical and limiting step for molecular assays, which prompted the arrival in Colombia of automated purification instruments during the SARS-CoV-2 pandemic. The local application of this technology in the study of tropical diseases, such as dengue and zika, is beginning to be tested. We evaluated the efficiency of the automated extraction of viral RNA for studies of pediatric dengue and zika. Clinical samples of children with dengue that were well characterized through RNA isolation by silica columns and serotype-specific nested RT-PCR (DENV-1 n = 7, DENV-2 n = 5, and negatives n = 8) in addition to 40 pediatric plasma samples spiked with ZIKV (strain PRVA BC59) and 209 from negative pre-epidemic children were analyzed. RNA from patients was extracted by two automated standard and high-throughput protocols on the KingFisher™ Flex instrument. The isolated RNA was evaluated for concentration and purity by spectrophotometry, for structural and functional integrity by electrophoresis and expression of the RNase P gene, and usefulness in serotype-specific DENV detection by conventional and real-time RT-PCR. For the evaluation of ZIKV RNA, the commercial TaqMan Triplex® assay was used, along with a well-tested in-house RT-qPCR assay. The concentration of RNA (5.2 vs. 7.5 ng/μL, P=0.03) and the number of integral bands (9 vs. 11) were higher with the high-throughput protocol. However, the number of specimens serotyped for DENV by RT-qPCR was comparable for both protocols. The cycle thresholds of the TaqMan Triplex® commercial kit and the in-house assay for the detection of plasma ZIKV RNA isolated with the standard protocol showed a strong association (r = 0.93, P<0.0001) and a Cohen Kappa index of 0.98 when all 249 samples were analyzed. These preliminary results suggest that automated instruments could be used in studies of cocirculating flaviviruses that have represented a public health problem in recent decades in Colombia. They boast advantages such as efficiency, precision, ... |
format |
Article in Journal/Newspaper |
author |
Sandra L. Delgado Piedad M. Perilla Doris M. Salgado María Clemencia Rojas Carlos F. Narváez |
author_facet |
Sandra L. Delgado Piedad M. Perilla Doris M. Salgado María Clemencia Rojas Carlos F. Narváez |
author_sort |
Sandra L. Delgado |
title |
Efficiency of Automated Viral RNA Purification for Pediatric Studies of Dengue and Zika in Hyperendemic Areas |
title_short |
Efficiency of Automated Viral RNA Purification for Pediatric Studies of Dengue and Zika in Hyperendemic Areas |
title_full |
Efficiency of Automated Viral RNA Purification for Pediatric Studies of Dengue and Zika in Hyperendemic Areas |
title_fullStr |
Efficiency of Automated Viral RNA Purification for Pediatric Studies of Dengue and Zika in Hyperendemic Areas |
title_full_unstemmed |
Efficiency of Automated Viral RNA Purification for Pediatric Studies of Dengue and Zika in Hyperendemic Areas |
title_sort |
efficiency of automated viral rna purification for pediatric studies of dengue and zika in hyperendemic areas |
publisher |
Hindawi Limited |
publishDate |
2023 |
url |
https://doi.org/10.1155/2023/1576481 https://doaj.org/article/597622845a2c461dbbaa41627f5978b8 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
Journal of Tropical Medicine, Vol 2023 (2023) |
op_relation |
http://dx.doi.org/10.1155/2023/1576481 https://doaj.org/toc/1687-9694 1687-9694 doi:10.1155/2023/1576481 https://doaj.org/article/597622845a2c461dbbaa41627f5978b8 |
op_doi |
https://doi.org/10.1155/2023/1576481 |
container_title |
Journal of Tropical Medicine |
container_volume |
2023 |
container_start_page |
1 |
op_container_end_page |
11 |
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1782331771148304384 |