Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis
Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved..
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and quickly spread around the world, forcing global health authorities to develop protocols for its diagnosis. Here we report dimer formation in the N2 primers-probe set (CDC 2019-nCoV Real-Time RT-PCR) used in the diagnostic routine, and propose alternatives to reduce dimerization events. Late unspecific amplifications were visualized in 56.4% of negative samples and 57.1% of no-template control, but not in positive samples or positive control. In silico analysis and gel electrophoresis confirmed the dimer formation. The RT-qPCR parameters were optimized and the late unspecific amplifications decreased to 11.5% in negative samples and no-template control. The adjustment of PCR parameters was essential to reduce the risk of false-positives results and to avoid inclusive results requiring repeat testing, which increases the costs and generates delays in results or even unnecessary requests for new samples.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:102 |
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| Enthalten in: |
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases - 102(2021) vom: 15. Jan., Seite 437-439 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Jaeger, Lauren Hubert [VerfasserIn] |
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| Links: |
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| Themen: |
COVID-19 |
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| Anmerkungen: |
Date Completed 14.01.2021 Date Revised 14.04.2021 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.ijid.2020.10.079 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM316986518 |
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| 520 | |a Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved. | ||
| 520 | |a Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and quickly spread around the world, forcing global health authorities to develop protocols for its diagnosis. Here we report dimer formation in the N2 primers-probe set (CDC 2019-nCoV Real-Time RT-PCR) used in the diagnostic routine, and propose alternatives to reduce dimerization events. Late unspecific amplifications were visualized in 56.4% of negative samples and 57.1% of no-template control, but not in positive samples or positive control. In silico analysis and gel electrophoresis confirmed the dimer formation. The RT-qPCR parameters were optimized and the late unspecific amplifications decreased to 11.5% in negative samples and no-template control. The adjustment of PCR parameters was essential to reduce the risk of false-positives results and to avoid inclusive results requiring repeat testing, which increases the costs and generates delays in results or even unnecessary requests for new samples | ||
| 650 | 4 | |a Case Reports | |
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| 700 | 1 | |a Duque, Ana Paula do Nascimento |e verfasserin |4 aut | |
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