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Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis
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...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598553/ https://www.ncbi.nlm.nih.gov/pubmed/33130201 http://dx.doi.org/10.1016/j.ijid.2020.10.079 |
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author | Jaeger, Lauren Hubert Nascimento, Thiago César Rocha, Fabíola Dutra Vilela, Fernanda Maria Pinto Duque, Ana Paula do Nascimento Silva, Lívia Mara Riani, Lorena Rodrigues Moreira, João Paulo Chagas, Jéssica Mara de Assis Pereira, Thamiris Vilela Perches, Carmen Gomide Pinto Watanabe, Aripuana Sakurada Aranha Viccini, Lyderson Facio Silvério, Marcelo Silva Corrêa, José Otávio do Amaral Pereira-Junior, Olavo dos Santos Pittella, Frederico |
author_facet | Jaeger, Lauren Hubert Nascimento, Thiago César Rocha, Fabíola Dutra Vilela, Fernanda Maria Pinto Duque, Ana Paula do Nascimento Silva, Lívia Mara Riani, Lorena Rodrigues Moreira, João Paulo Chagas, Jéssica Mara de Assis Pereira, Thamiris Vilela Perches, Carmen Gomide Pinto Watanabe, Aripuana Sakurada Aranha Viccini, Lyderson Facio Silvério, Marcelo Silva Corrêa, José Otávio do Amaral Pereira-Junior, Olavo dos Santos Pittella, Frederico |
author_sort | Jaeger, Lauren Hubert |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7598553 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. |
record_format | MEDLINE/PubMed |
spelling | pubmed-75985532020-11-02 Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis Jaeger, Lauren Hubert Nascimento, Thiago César Rocha, Fabíola Dutra Vilela, Fernanda Maria Pinto Duque, Ana Paula do Nascimento Silva, Lívia Mara Riani, Lorena Rodrigues Moreira, João Paulo Chagas, Jéssica Mara de Assis Pereira, Thamiris Vilela Perches, Carmen Gomide Pinto Watanabe, Aripuana Sakurada Aranha Viccini, Lyderson Facio Silvério, Marcelo Silva Corrêa, José Otávio do Amaral Pereira-Junior, Olavo dos Santos Pittella, Frederico Int J Infect Dis Case Report 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. The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. 2021-01 2020-10-29 /pmc/articles/PMC7598553/ /pubmed/33130201 http://dx.doi.org/10.1016/j.ijid.2020.10.079 Text en © 2020 The Authors Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. |
spellingShingle | Case Report Jaeger, Lauren Hubert Nascimento, Thiago César Rocha, Fabíola Dutra Vilela, Fernanda Maria Pinto Duque, Ana Paula do Nascimento Silva, Lívia Mara Riani, Lorena Rodrigues Moreira, João Paulo Chagas, Jéssica Mara de Assis Pereira, Thamiris Vilela Perches, Carmen Gomide Pinto Watanabe, Aripuana Sakurada Aranha Viccini, Lyderson Facio Silvério, Marcelo Silva Corrêa, José Otávio do Amaral Pereira-Junior, Olavo dos Santos Pittella, Frederico Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis |
title | Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis |
title_full | Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis |
title_fullStr | Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis |
title_full_unstemmed | Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis |
title_short | Adjusting RT-qPCR conditions to avoid unspecific amplification in SARS-CoV-2 diagnosis |
title_sort | adjusting rt-qpcr conditions to avoid unspecific amplification in sars-cov-2 diagnosis |
topic | Case Report |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598553/ https://www.ncbi.nlm.nih.gov/pubmed/33130201 http://dx.doi.org/10.1016/j.ijid.2020.10.079 |
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