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Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests

Bindu Swapna Madala, Andre L. M. Reis, Ira W. Deveson, William Rawlinson, Tim R. Mercer
doi: https://doi.org/10.1101/2020.06.09.143412
Bindu Swapna Madala
1Garvan Institute of Medical Research, NSW, Australia
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Andre L. M. Reis
1Garvan Institute of Medical Research, NSW, Australia
2St Vincent’s Clinical School, University of New South Wales, NSW, Australia
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Ira W. Deveson
1Garvan Institute of Medical Research, NSW, Australia
2St Vincent’s Clinical School, University of New South Wales, NSW, Australia
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William Rawlinson
3School of Medical Sciences, Women’s & Children’s, and School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia
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Tim R. Mercer
1Garvan Institute of Medical Research, NSW, Australia
2St Vincent’s Clinical School, University of New South Wales, NSW, Australia
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  • For correspondence: t.mercer@garvan.org.au
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ABSTRACT

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 Centers 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.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted June 11, 2020.
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Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests
Bindu Swapna Madala, Andre L. M. Reis, Ira W. Deveson, William Rawlinson, Tim R. Mercer
bioRxiv 2020.06.09.143412; doi: https://doi.org/10.1101/2020.06.09.143412
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Chimeric synthetic reference standards enable cross-validation of positive and negative controls in SARS-CoV-2 molecular tests
Bindu Swapna Madala, Andre L. M. Reis, Ira W. Deveson, William Rawlinson, Tim R. Mercer
bioRxiv 2020.06.09.143412; doi: https://doi.org/10.1101/2020.06.09.143412

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