Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Revista da Sociedade Brasileira de Medicina Tropical |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822017000300350 |
Resumo: | Abstract INTRODUCTION: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol developed for the simultaneous detection of Leishmania infantum DNA and sample quality control. METHODS: After preliminary tests with the newly designed TaqMan® probes for the two targets ( L. infantum and glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene), the duplex qPCR protocol was optimized. For the evaluation of the standardized protocol, human blood samples were tested (n=68) and the results were compared to those obtained by reference diagnostic techniques. Statistical analyses included percentage agreement and the Kappa ( k ) coefficient. RESULTS: The detection limit of L. infantum DNA reached 2x10 2 fg (corresponding to ~1 parasite) per µL of blood (ε: 93.9%). The percentage agreement obtained between the duplex VL qPCR and the reference techniques was individually obtained as follows: molecular: 88.3% ( k =0.666; 95% CI 0.437-0.894, good), and serological: 81.7% ( k =0.411; 95% CI 0.125-0.697, moderate). Between the reference techniques, the percentage agreement was 86.7% ( k =0.586; 95% CI 0.332-0.840, moderate). CONCLUSIONS: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers. |
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Revista da Sociedade Brasileira de Medicina Tropical |
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Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approachVisceral leishmaniasisDiagnosisHIV/VL co-infectionSample quality controlDuplex qPCRAbstract INTRODUCTION: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol developed for the simultaneous detection of Leishmania infantum DNA and sample quality control. METHODS: After preliminary tests with the newly designed TaqMan® probes for the two targets ( L. infantum and glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene), the duplex qPCR protocol was optimized. For the evaluation of the standardized protocol, human blood samples were tested (n=68) and the results were compared to those obtained by reference diagnostic techniques. Statistical analyses included percentage agreement and the Kappa ( k ) coefficient. RESULTS: The detection limit of L. infantum DNA reached 2x10 2 fg (corresponding to ~1 parasite) per µL of blood (ε: 93.9%). The percentage agreement obtained between the duplex VL qPCR and the reference techniques was individually obtained as follows: molecular: 88.3% ( k =0.666; 95% CI 0.437-0.894, good), and serological: 81.7% ( k =0.411; 95% CI 0.125-0.697, moderate). Between the reference techniques, the percentage agreement was 86.7% ( k =0.586; 95% CI 0.332-0.840, moderate). CONCLUSIONS: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers.Sociedade Brasileira de Medicina Tropical - SBMT2017-06-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822017000300350Revista da Sociedade Brasileira de Medicina Tropical v.50 n.3 2017reponame:Revista da Sociedade Brasileira de Medicina Tropicalinstname:Sociedade Brasileira de Medicina Tropical (SBMT)instacron:SBMT10.1590/0037-8682-0012-2017info:eu-repo/semantics/openAccessTrajano-Silva,Lays Adrianne MendonçaPessoa-e-Silva,RômuloGonçalves-de-Albuquerque,Suênia da CunhaMorais,Rayana Carla Silva deCosta-Oliveira,Cíntia Nascimento daGoes,Tayná Correia dePaiva-Cavalcanti,Milena deeng2017-09-12T00:00:00Zoai:scielo:S0037-86822017000300350Revistahttps://www.sbmt.org.br/portal/revista/ONGhttps://old.scielo.br/oai/scielo-oai.php||dalmo@rsbmt.uftm.edu.br|| rsbmt@rsbmt.uftm.edu.br1678-98490037-8682opendoar:2017-09-12T00:00Revista da Sociedade Brasileira de Medicina Tropical - Sociedade Brasileira de Medicina Tropical (SBMT)false |
dc.title.none.fl_str_mv |
Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
title |
Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
spellingShingle |
Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach Trajano-Silva,Lays Adrianne Mendonça Visceral leishmaniasis Diagnosis HIV/VL co-infection Sample quality control Duplex qPCR |
title_short |
Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
title_full |
Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
title_fullStr |
Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
title_full_unstemmed |
Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
title_sort |
Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
author |
Trajano-Silva,Lays Adrianne Mendonça |
author_facet |
Trajano-Silva,Lays Adrianne Mendonça Pessoa-e-Silva,Rômulo Gonçalves-de-Albuquerque,Suênia da Cunha Morais,Rayana Carla Silva de Costa-Oliveira,Cíntia Nascimento da Goes,Tayná Correia de Paiva-Cavalcanti,Milena de |
author_role |
author |
author2 |
Pessoa-e-Silva,Rômulo Gonçalves-de-Albuquerque,Suênia da Cunha Morais,Rayana Carla Silva de Costa-Oliveira,Cíntia Nascimento da Goes,Tayná Correia de Paiva-Cavalcanti,Milena de |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Trajano-Silva,Lays Adrianne Mendonça Pessoa-e-Silva,Rômulo Gonçalves-de-Albuquerque,Suênia da Cunha Morais,Rayana Carla Silva de Costa-Oliveira,Cíntia Nascimento da Goes,Tayná Correia de Paiva-Cavalcanti,Milena de |
dc.subject.por.fl_str_mv |
Visceral leishmaniasis Diagnosis HIV/VL co-infection Sample quality control Duplex qPCR |
topic |
Visceral leishmaniasis Diagnosis HIV/VL co-infection Sample quality control Duplex qPCR |
description |
Abstract INTRODUCTION: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol developed for the simultaneous detection of Leishmania infantum DNA and sample quality control. METHODS: After preliminary tests with the newly designed TaqMan® probes for the two targets ( L. infantum and glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene), the duplex qPCR protocol was optimized. For the evaluation of the standardized protocol, human blood samples were tested (n=68) and the results were compared to those obtained by reference diagnostic techniques. Statistical analyses included percentage agreement and the Kappa ( k ) coefficient. RESULTS: The detection limit of L. infantum DNA reached 2x10 2 fg (corresponding to ~1 parasite) per µL of blood (ε: 93.9%). The percentage agreement obtained between the duplex VL qPCR and the reference techniques was individually obtained as follows: molecular: 88.3% ( k =0.666; 95% CI 0.437-0.894, good), and serological: 81.7% ( k =0.411; 95% CI 0.125-0.697, moderate). Between the reference techniques, the percentage agreement was 86.7% ( k =0.586; 95% CI 0.332-0.840, moderate). CONCLUSIONS: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers. |
publishDate |
2017 |
dc.date.none.fl_str_mv |
2017-06-01 |
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://old.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822017000300350 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822017000300350 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/0037-8682-0012-2017 |
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 |
Sociedade Brasileira de Medicina Tropical - SBMT |
publisher.none.fl_str_mv |
Sociedade Brasileira de Medicina Tropical - SBMT |
dc.source.none.fl_str_mv |
Revista da Sociedade Brasileira de Medicina Tropical v.50 n.3 2017 reponame:Revista da Sociedade Brasileira de Medicina Tropical instname:Sociedade Brasileira de Medicina Tropical (SBMT) instacron:SBMT |
instname_str |
Sociedade Brasileira de Medicina Tropical (SBMT) |
instacron_str |
SBMT |
institution |
SBMT |
reponame_str |
Revista da Sociedade Brasileira de Medicina Tropical |
collection |
Revista da Sociedade Brasileira de Medicina Tropical |
repository.name.fl_str_mv |
Revista da Sociedade Brasileira de Medicina Tropical - Sociedade Brasileira de Medicina Tropical (SBMT) |
repository.mail.fl_str_mv |
||dalmo@rsbmt.uftm.edu.br|| rsbmt@rsbmt.uftm.edu.br |
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1752122160665591808 |