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A Novel, Reverse Transcription, Droplet Digital PCR Assay for the Combined, Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus 2 with Swine Acute Diarrhea Syndrome Coronavirus.
- Source :
-
Journal of AOAC International [J AOAC Int] 2022 Sep 06; Vol. 105 (5), pp. 1437-1446. - Publication Year :
- 2022
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Abstract
- Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread over the world since its emergence. Although the dominant route of SARS-CoV-2 infection is respiratory, a number of studies revealed infection risk from contaminated surfaces and products, including porcine-derived food and other products. The SARS-CoV-2 outbreak has been severely threatening public health, and disrupting porcine products trade and the pig industry. Swine acute diarrhea syndrome coronavirus (SADS-CoV), which was responsible for large-scale, fatal disease in piglets, emerged in 2017 and has caused enormous economic losses in the pig industry. Currently, reverse transcription real-time PCR (RT-rPCR) is the gold standard method for SARS-CoV-2 diagnosis and is most commonly used for SADS-CoV detection. However, inaccurate detection of the SARS-CoV-2 infection obtained by RT-rPCR is increasingly reported, especially in specimens with low viral load.<br />Objective: This study aimed to develop an accurate reverse transcription droplet digital PCR (RT-ddPCR) assay for the detection of SARS-CoV-2 and SADS-CoV simultaneously.<br />Methods: Two pairs of primers and one double-quenched probe targeting the RNA-dependent RNA polymerase (RDRP) region of the open reading frame 1ab (ORF1ab) gene of SARS-CoV-2 and the corresponding ORF1ab region of SADS-CoV were designed to develop the RT-ddPCR assay. The sensitivity, specificity, repeatability, and reproducibility were tested using complementary RNAs (cRNAs) and clinical specimens.<br />Results: The detection limits of RT-ddPCR were 1.48 ± 0.18 and 1.38 ± 0.17 copies in a 20 μL reaction for SARS-CoV-2 and SADS-CoV cRNAs, respectively (n = 8), showing approximately 4- and 10-fold greater sensitivity than the RT-rPCR assay. This assay also exhibited good specificity, repeatability, and reproducibility.<br />Conclusion: The established RT-ddPCR assay was shown to be a highly effective, accurate, and reliable method for the sensitive detection of SARS-CoV-2 and SADS-CoV.<br />Highlights: This RT-ddPCR assay could be used to detect both SARS-CoV-2 and SADS-CoV in a sample with one double-quenched probe, and is also the first reported RT-ddPCR assay for SADS-CoV detection.<br /> (© The Author(s) 2022. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
Details
- Language :
- English
- ISSN :
- 1944-7922
- Volume :
- 105
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Journal of AOAC International
- Publication Type :
- Academic Journal
- Accession number :
- 35377440
- Full Text :
- https://doi.org/10.1093/jaoacint/qsac039