Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Outros Autores: | , , , , , |
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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Memórias do Instituto Oswaldo Cruz |
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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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1669937727177490432 |