Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria

Detalhes bibliográficos
Autor(a) principal: Almeida-de-Oliveira,Natália Ketrin
Data de Publicação: 2019
Outros Autores: Moreira,Otacílio C, de Lavigne,Aline Rosa, Mendonça-Lima,Leila, Werneck,Guilherme Loureiro, Daniel-Ribeiro,Cláudio Tadeu, Ferreira-da-Cruz,Maria de Fátima
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Memórias do Instituto Oswaldo Cruz
Texto Completo: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762019000100308
Resumo: BACKGROUND The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination. OBJECTIVE Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy. METHODS The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision. FINDINGS The linearity in SYBR™ Green and TaqMan™ systems was 106 and 102 copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR. CONCLUSION Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors.
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spelling Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malariamalariadiagnosisPlasmodium vivax BACKGROUND The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination. OBJECTIVE Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy. METHODS The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision. FINDINGS The linearity in SYBR™ Green and TaqMan™ systems was 106 and 102 copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR. CONCLUSION Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors.Instituto Oswaldo Cruz, Ministério da Saúde2019-01-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762019000100308Memórias do Instituto Oswaldo Cruz v.114 2019reponame:Memórias do Instituto Oswaldo Cruzinstname:Fundação Oswaldo Cruzinstacron:FIOCRUZ10.1590/0074-02760180350info:eu-repo/semantics/openAccessAlmeida-de-Oliveira,Natália KetrinMoreira,Otacílio Cde Lavigne,Aline RosaMendonça-Lima,LeilaWerneck,Guilherme LoureiroDaniel-Ribeiro,Cláudio TadeuFerreira-da-Cruz,Maria de Fátimaeng2020-04-25T17:52:57Zhttp://www.scielo.br/oai/scielo-oai.php0074-02761678-8060opendoar:null2020-04-26 02:22:30.409Memórias do Instituto Oswaldo Cruz - Fundação Oswaldo Cruztrue
dc.title.none.fl_str_mv Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
title Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
spellingShingle Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
Almeida-de-Oliveira,Natália Ketrin
malaria
diagnosis
Plasmodium vivax
title_short Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
title_full Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
title_fullStr Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
title_full_unstemmed Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
title_sort Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
author Almeida-de-Oliveira,Natália Ketrin
author_facet Almeida-de-Oliveira,Natália Ketrin
Moreira,Otacílio C
de Lavigne,Aline Rosa
Mendonça-Lima,Leila
Werneck,Guilherme Loureiro
Daniel-Ribeiro,Cláudio Tadeu
Ferreira-da-Cruz,Maria de Fátima
author_role author
author2 Moreira,Otacílio C
de Lavigne,Aline Rosa
Mendonça-Lima,Leila
Werneck,Guilherme Loureiro
Daniel-Ribeiro,Cláudio Tadeu
Ferreira-da-Cruz,Maria de Fátima
author2_role author
author
author
author
author
author
dc.contributor.author.fl_str_mv Almeida-de-Oliveira,Natália Ketrin
Moreira,Otacílio C
de Lavigne,Aline Rosa
Mendonça-Lima,Leila
Werneck,Guilherme Loureiro
Daniel-Ribeiro,Cláudio Tadeu
Ferreira-da-Cruz,Maria de Fátima
dc.subject.por.fl_str_mv malaria
diagnosis
Plasmodium vivax
topic malaria
diagnosis
Plasmodium vivax
dc.description.none.fl_txt_mv BACKGROUND The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination. OBJECTIVE Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy. METHODS The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision. FINDINGS The linearity in SYBR™ Green and TaqMan™ systems was 106 and 102 copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR. CONCLUSION Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors.
description BACKGROUND The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination. OBJECTIVE Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy. METHODS The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision. FINDINGS The linearity in SYBR™ Green and TaqMan™ systems was 106 and 102 copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR. CONCLUSION Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors.
publishDate 2019
dc.date.none.fl_str_mv 2019-01-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762019000100308
url http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762019000100308
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1590/0074-02760180350
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv text/html
dc.publisher.none.fl_str_mv Instituto Oswaldo Cruz, Ministério da Saúde
publisher.none.fl_str_mv Instituto Oswaldo Cruz, Ministério da Saúde
dc.source.none.fl_str_mv Memórias do Instituto Oswaldo Cruz v.114 2019
reponame:Memórias do Instituto Oswaldo Cruz
instname:Fundação Oswaldo Cruz
instacron:FIOCRUZ
reponame_str Memórias do Instituto Oswaldo Cruz
collection Memórias do Instituto Oswaldo Cruz
instname_str Fundação Oswaldo Cruz
instacron_str FIOCRUZ
institution FIOCRUZ
repository.name.fl_str_mv Memórias do Instituto Oswaldo Cruz - Fundação Oswaldo Cruz
repository.mail.fl_str_mv
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