Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load
BACKGROUND Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection. OBJECTIVES In this study, we developed an open-source RNA standar...
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| Format: | Article |
| Language: | English |
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Fundação Oswaldo Cruz (FIOCRUZ)
2022-01-01
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| Series: | Memorias do Instituto Oswaldo Cruz |
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| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762021000101108&tlng=en |
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| author | Juliana Comerlato Carolina Baldisserotto Comerlato Fernando Hayashi Sant’Anna Marina Bessel Celina Monteiro Abreu Eliana Márcia Wendland |
| author_facet | Juliana Comerlato Carolina Baldisserotto Comerlato Fernando Hayashi Sant’Anna Marina Bessel Celina Monteiro Abreu Eliana Márcia Wendland |
| author_sort | Juliana Comerlato |
| collection | DOAJ |
| description | BACKGROUND Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection. OBJECTIVES In this study, we developed an open-source RNA standard-based real-time quantitative RT-PCR (RT-qPCR) assay for quantitative diagnostics of SARS-CoV-2 from nasopharynx, oropharynx, saliva and plasma samples. METHODS AND FINDINGS We evaluated three SARS-CoV-2 target genes and selected the RNA-dependent RNA polymerase (RdRp) gene, given its better performance. To improve the efficiency of the assay, a primer gradient containing 25 primers forward and reverse concentration combinations was performed. The forward and reverse primer pairs with 400 nM and 500 nM concentrations, respectively, showed the highest sensitivity. The LOD95% was ~60 copies per reaction. From the four biological matrices tested, none of them interfered with the viral load measurement. Comparison with the AllplexTM 2019-nCoV assay (Seegene) demonstrated that our test presents 90% sensitivity and 100% specificity. MAIN CONCLUSIONS We developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples. |
| first_indexed | 2024-03-12T09:31:23Z |
| format | Article |
| id | doaj.art-a1a3339dbf074da5ae1e680b11359ff8 |
| institution | Directory Open Access Journal |
| issn | 1678-8060 |
| language | English |
| last_indexed | 2024-03-12T09:31:23Z |
| publishDate | 2022-01-01 |
| publisher | Fundação Oswaldo Cruz (FIOCRUZ) |
| record_format | Article |
| series | Memorias do Instituto Oswaldo Cruz |
| spelling | doaj.art-a1a3339dbf074da5ae1e680b11359ff82023-09-02T13:54:13ZengFundação Oswaldo Cruz (FIOCRUZ)Memorias do Instituto Oswaldo Cruz1678-80602022-01-0111610.1590/0074-02760210237Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral loadJuliana ComerlatoCarolina Baldisserotto ComerlatoFernando Hayashi Sant’AnnaMarina BesselCelina Monteiro AbreuEliana Márcia Wendlandhttps://orcid.org/0000-0001-6356-6413BACKGROUND Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection. OBJECTIVES In this study, we developed an open-source RNA standard-based real-time quantitative RT-PCR (RT-qPCR) assay for quantitative diagnostics of SARS-CoV-2 from nasopharynx, oropharynx, saliva and plasma samples. METHODS AND FINDINGS We evaluated three SARS-CoV-2 target genes and selected the RNA-dependent RNA polymerase (RdRp) gene, given its better performance. To improve the efficiency of the assay, a primer gradient containing 25 primers forward and reverse concentration combinations was performed. The forward and reverse primer pairs with 400 nM and 500 nM concentrations, respectively, showed the highest sensitivity. The LOD95% was ~60 copies per reaction. From the four biological matrices tested, none of them interfered with the viral load measurement. Comparison with the AllplexTM 2019-nCoV assay (Seegene) demonstrated that our test presents 90% sensitivity and 100% specificity. MAIN CONCLUSIONS We developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762021000101108&tlng=enreverse transcriptase polymerase chain reactionreal-time polymerase chain reactionSARS-CoV-2COVID-19viral loadCOVID-19 testing |
| spellingShingle | Juliana Comerlato Carolina Baldisserotto Comerlato Fernando Hayashi Sant’Anna Marina Bessel Celina Monteiro Abreu Eliana Márcia Wendland Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load Memorias do Instituto Oswaldo Cruz reverse transcriptase polymerase chain reaction real-time polymerase chain reaction SARS-CoV-2 COVID-19 viral load COVID-19 testing |
| title | Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load |
| title_full | Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load |
| title_fullStr | Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load |
| title_full_unstemmed | Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load |
| title_short | Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load |
| title_sort | open source real time quantitative rt pcr based on a rna standard for the assessment of sars cov 2 viral load |
| topic | reverse transcriptase polymerase chain reaction real-time polymerase chain reaction SARS-CoV-2 COVID-19 viral load COVID-19 testing |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762021000101108&tlng=en |
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