COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols
ABSTRACT The gold standard for the laboratory diagnosis of COVID-19 is the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay, which searches for SARS-CoV-2 target genes in nasopharyngeal/oropharyngeal (NP/OP) samples, and its performance depends on the quantity a...
Published in: | Revista do Instituto de Medicina Tropical de São Paulo |
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Main Authors: | , , , , , , |
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Language: | English |
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Universidade de São Paulo (USP)
2021
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Online Access: | https://doi.org/10.1590/s1678-9946202163052 https://doaj.org/article/7a62c2321a9f4165a5366ea6369a6192 |
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ftdoajarticles:oai:doaj.org/article:7a62c2321a9f4165a5366ea6369a6192 2024-09-09T19:28:26+00:00 COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols Karoline Rodrigues Campos Cláudio Tavares Sacchi Cláudia Regina Gonçalves Érica Valessa Ramos Gomes Pagnoca Alana dos Santos Dias Lucila Okuyama Fukasawa Adele Caterino-de-Araujo 2021-06-01T00:00:00Z https://doi.org/10.1590/s1678-9946202163052 https://doaj.org/article/7a62c2321a9f4165a5366ea6369a6192 EN eng Universidade de São Paulo (USP) http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0036-46652021000100228&tlng=en https://doaj.org/toc/1678-9946 1678-9946 doi:10.1590/s1678-9946202163052 https://doaj.org/article/7a62c2321a9f4165a5366ea6369a6192 Revista do Instituto de Medicina Tropical de São Paulo, Vol 63 (2021) COVID-19 SARS-CoV-2 SARS-CoV-2 RT-PCR SARS-CoV-2 molecular diagnosis RNA extraction methods Assays performances Cost-effectiveness RT-qPCR Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 article 2021 ftdoajarticles https://doi.org/10.1590/s1678-9946202163052 2024-08-05T17:49:31Z ABSTRACT The gold standard for the laboratory diagnosis of COVID-19 is the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay, which searches for SARS-CoV-2 target genes in nasopharyngeal/oropharyngeal (NP/OP) samples, and its performance depends on the quantity and quality of the RNA input. This study compared the performance and cost-effectiveness of three different kits/reagents for RNA extraction used in COVID-19 diagnosis in Sao Paulo, Brazil. A total of 300 NP/OP samples belonging to suspected cases of COVID-19 stored in a biorepository were randomly selected, and RNA was extracted using (i) automated extraction (Loccus, Extracta Kit FAST), (ii) manual extraction (BioGene Kit, Bioclin, Quibasa), and (iii) quick extraction methods (Lucigen, Quick DNA Extract Kit). Next, the samples were tested using RT-qPCR for SARS-CoV-2 with the Allplex 2019-nCoV modified assay and the Charité-Berlin protocol. All assays/kits were used according to the manufacturer’s instructions. For the Allplex kit, the sensitivity in detecting SARS-CoV-2 with previously extracted RNA by different procedures was 100.0% for Loccus, 100.0% for BioGene and 91.9% for Quick. Using the Charité-Berlin protocol, the sensitivities were 81.4% for Loccus, 81.2% for BioGene and 60.7% for Quick. The least sensitive target gene and the gene most affected by RNA extraction procedures was the RNA-dependent RNA polymerase gene (Charité-Berlin protocol). No false-positive SARS-CoV-2 results were detected using RNA obtained from any of the different protocols. In conclusion, Loccus and BioGene RNA extractions were efficient for RT-qPCR assays, and although the BioGene procedure is less expensive, Loccus is the best choice because it allows the rapid handling of hundreds or thousands of samples, a desirable feature during pandemics. Although less sensitive, the Quick extraction is useful during outbreaks coupled with the Allplex amplification kit for SARS-CoV-2 diagnosis (κ = 0.925). Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Revista do Instituto de Medicina Tropical de São Paulo 63 |
institution |
Open Polar |
collection |
Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
COVID-19 SARS-CoV-2 SARS-CoV-2 RT-PCR SARS-CoV-2 molecular diagnosis RNA extraction methods Assays performances Cost-effectiveness RT-qPCR Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
spellingShingle |
COVID-19 SARS-CoV-2 SARS-CoV-2 RT-PCR SARS-CoV-2 molecular diagnosis RNA extraction methods Assays performances Cost-effectiveness RT-qPCR Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 Karoline Rodrigues Campos Cláudio Tavares Sacchi Cláudia Regina Gonçalves Érica Valessa Ramos Gomes Pagnoca Alana dos Santos Dias Lucila Okuyama Fukasawa Adele Caterino-de-Araujo COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols |
topic_facet |
COVID-19 SARS-CoV-2 SARS-CoV-2 RT-PCR SARS-CoV-2 molecular diagnosis RNA extraction methods Assays performances Cost-effectiveness RT-qPCR Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
description |
ABSTRACT The gold standard for the laboratory diagnosis of COVID-19 is the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) assay, which searches for SARS-CoV-2 target genes in nasopharyngeal/oropharyngeal (NP/OP) samples, and its performance depends on the quantity and quality of the RNA input. This study compared the performance and cost-effectiveness of three different kits/reagents for RNA extraction used in COVID-19 diagnosis in Sao Paulo, Brazil. A total of 300 NP/OP samples belonging to suspected cases of COVID-19 stored in a biorepository were randomly selected, and RNA was extracted using (i) automated extraction (Loccus, Extracta Kit FAST), (ii) manual extraction (BioGene Kit, Bioclin, Quibasa), and (iii) quick extraction methods (Lucigen, Quick DNA Extract Kit). Next, the samples were tested using RT-qPCR for SARS-CoV-2 with the Allplex 2019-nCoV modified assay and the Charité-Berlin protocol. All assays/kits were used according to the manufacturer’s instructions. For the Allplex kit, the sensitivity in detecting SARS-CoV-2 with previously extracted RNA by different procedures was 100.0% for Loccus, 100.0% for BioGene and 91.9% for Quick. Using the Charité-Berlin protocol, the sensitivities were 81.4% for Loccus, 81.2% for BioGene and 60.7% for Quick. The least sensitive target gene and the gene most affected by RNA extraction procedures was the RNA-dependent RNA polymerase gene (Charité-Berlin protocol). No false-positive SARS-CoV-2 results were detected using RNA obtained from any of the different protocols. In conclusion, Loccus and BioGene RNA extractions were efficient for RT-qPCR assays, and although the BioGene procedure is less expensive, Loccus is the best choice because it allows the rapid handling of hundreds or thousands of samples, a desirable feature during pandemics. Although less sensitive, the Quick extraction is useful during outbreaks coupled with the Allplex amplification kit for SARS-CoV-2 diagnosis (κ = 0.925). |
format |
Article in Journal/Newspaper |
author |
Karoline Rodrigues Campos Cláudio Tavares Sacchi Cláudia Regina Gonçalves Érica Valessa Ramos Gomes Pagnoca Alana dos Santos Dias Lucila Okuyama Fukasawa Adele Caterino-de-Araujo |
author_facet |
Karoline Rodrigues Campos Cláudio Tavares Sacchi Cláudia Regina Gonçalves Érica Valessa Ramos Gomes Pagnoca Alana dos Santos Dias Lucila Okuyama Fukasawa Adele Caterino-de-Araujo |
author_sort |
Karoline Rodrigues Campos |
title |
COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols |
title_short |
COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols |
title_full |
COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols |
title_fullStr |
COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols |
title_full_unstemmed |
COVID-19 laboratory diagnosis: comparative analysis of different RNA extraction methods for SARS-CoV-2 detection by two amplification protocols |
title_sort |
covid-19 laboratory diagnosis: comparative analysis of different rna extraction methods for sars-cov-2 detection by two amplification protocols |
publisher |
Universidade de São Paulo (USP) |
publishDate |
2021 |
url |
https://doi.org/10.1590/s1678-9946202163052 https://doaj.org/article/7a62c2321a9f4165a5366ea6369a6192 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
Revista do Instituto de Medicina Tropical de São Paulo, Vol 63 (2021) |
op_relation |
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0036-46652021000100228&tlng=en https://doaj.org/toc/1678-9946 1678-9946 doi:10.1590/s1678-9946202163052 https://doaj.org/article/7a62c2321a9f4165a5366ea6369a6192 |
op_doi |
https://doi.org/10.1590/s1678-9946202163052 |
container_title |
Revista do Instituto de Medicina Tropical de São Paulo |
container_volume |
63 |
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