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Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests
DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target genetic material, and act only as positive control...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846570/ https://www.ncbi.nlm.nih.gov/pubmed/33514761 http://dx.doi.org/10.1038/s41598-021-81760-0 |
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author | Madala, Bindu Swapna Reis, Andre L. M. Deveson, Ira W. Rawlinson, William Mercer, Tim R. |
author_facet | Madala, Bindu Swapna Reis, Andre L. M. Deveson, Ira W. Rawlinson, William Mercer, Tim R. |
author_sort | Madala, Bindu Swapna |
collection | PubMed |
description | DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. However, negative controls are also required to evaluate test specificity. Using a pair of chimeric A/B RNA standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The chimeric standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. This design enables cross-validation of positive and negative controls between the paired standards in the same reaction, with identical conditions. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. The chimeric A/B standards were assessed using the US Centres for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS-CoV-2 in patient samples. This chimeric reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms. |
format | Online Article Text |
id | pubmed-7846570 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-78465702021-02-01 Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests Madala, Bindu Swapna Reis, Andre L. M. Deveson, Ira W. Rawlinson, William Mercer, Tim R. Sci Rep Article DNA synthesis in vitro has enabled the rapid production of reference standards. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. However, negative controls are also required to evaluate test specificity. Using a pair of chimeric A/B RNA standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The chimeric standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. This design enables cross-validation of positive and negative controls between the paired standards in the same reaction, with identical conditions. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. The chimeric A/B standards were assessed using the US Centres for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS-CoV-2 in patient samples. This chimeric reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms. Nature Publishing Group UK 2021-01-29 /pmc/articles/PMC7846570/ /pubmed/33514761 http://dx.doi.org/10.1038/s41598-021-81760-0 Text en © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Madala, Bindu Swapna Reis, Andre L. M. Deveson, Ira W. Rawlinson, William Mercer, Tim R. Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests |
title | Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests |
title_full | Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests |
title_fullStr | Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests |
title_full_unstemmed | Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests |
title_short | Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests |
title_sort | chimeric synthetic reference standards enable cross-validation of positive and negative controls in sars-cov-2 molecular tests |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846570/ https://www.ncbi.nlm.nih.gov/pubmed/33514761 http://dx.doi.org/10.1038/s41598-021-81760-0 |
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