Analytical performance of Four Polymerase Chain Reaction (PCR) and real time PCR (qPCR) assays for the detection of six Leishmania species DNA in Colombia

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real tim...

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Autores Principales: León, Cielo M., Muñoz, Marina, Galindo-Hernández, Carolina, Ayala, Martha S., Flórez, Carolina, Teherán, Aníbal, Cubides, Juan R., Ramírez, Juan David
Formato: Artículo (Article)
Lenguaje:Inglés (English)
Publicado: Frontiers Media S.A. 2017
Materias:
Pcr
Acceso en línea:https://repository.urosario.edu.co/handle/10336/24094
https://doi.org/10.3389/fmicb.2017.01907
id ir-10336-24094
recordtype dspace
spelling ir-10336-240942022-05-02T12:37:16Z Analytical performance of Four Polymerase Chain Reaction (PCR) and real time PCR (qPCR) assays for the detection of six Leishmania species DNA in Colombia León, Cielo M. Muñoz, Marina Galindo-Hernández, Carolina Ayala, Martha S. Flórez, Carolina Teherán, Aníbal Cubides, Juan R. Ramírez, Juan David Heat shock protein 70 Kinetoplast dna Molecular marker Rna 18s Analytical parameters Anticipated reportable range Article Colombia Controlled study Dna extraction Gene amplification Leishmania Limit of detection Measurement accuracy Microorganism detection Nonhuman Polymerase chain reaction Post hoc analysis Quantitative analysis Real time polymerase chain reaction Reproducibility Sensitivity and specificity Analytical performance Leishmania Molecular diagnosis Pcr Qpcr Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America. © 2017 León, Muñoz, Hernández, Ayala, Flórez, Teherán, Cubides and Ramírez. 2017 2020-05-26T00:08:34Z info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 1664302X https://repository.urosario.edu.co/handle/10336/24094 https://doi.org/10.3389/fmicb.2017.01907 eng info:eu-repo/semantics/openAccess application/pdf Frontiers Media S.A. instname:Universidad del Rosario
institution EdocUR - Universidad del Rosario
collection DSpace
language Inglés (English)
topic Heat shock protein 70
Kinetoplast dna
Molecular marker
Rna 18s
Analytical parameters
Anticipated reportable range
Article
Colombia
Controlled study
Dna extraction
Gene amplification
Leishmania
Limit of detection
Measurement accuracy
Microorganism detection
Nonhuman
Polymerase chain reaction
Post hoc analysis
Quantitative analysis
Real time polymerase chain reaction
Reproducibility
Sensitivity and specificity
Analytical performance
Leishmania
Molecular diagnosis
Pcr
Qpcr
spellingShingle Heat shock protein 70
Kinetoplast dna
Molecular marker
Rna 18s
Analytical parameters
Anticipated reportable range
Article
Colombia
Controlled study
Dna extraction
Gene amplification
Leishmania
Limit of detection
Measurement accuracy
Microorganism detection
Nonhuman
Polymerase chain reaction
Post hoc analysis
Quantitative analysis
Real time polymerase chain reaction
Reproducibility
Sensitivity and specificity
Analytical performance
Leishmania
Molecular diagnosis
Pcr
Qpcr
León, Cielo M.
Muñoz, Marina
Galindo-Hernández, Carolina
Ayala, Martha S.
Flórez, Carolina
Teherán, Aníbal
Cubides, Juan R.
Ramírez, Juan David
Analytical performance of Four Polymerase Chain Reaction (PCR) and real time PCR (qPCR) assays for the detection of six Leishmania species DNA in Colombia
description Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America. © 2017 León, Muñoz, Hernández, Ayala, Flórez, Teherán, Cubides and Ramírez.
format Artículo (Article)
author León, Cielo M.
Muñoz, Marina
Galindo-Hernández, Carolina
Ayala, Martha S.
Flórez, Carolina
Teherán, Aníbal
Cubides, Juan R.
Ramírez, Juan David
author_facet León, Cielo M.
Muñoz, Marina
Galindo-Hernández, Carolina
Ayala, Martha S.
Flórez, Carolina
Teherán, Aníbal
Cubides, Juan R.
Ramírez, Juan David
author_sort León, Cielo M.
title Analytical performance of Four Polymerase Chain Reaction (PCR) and real time PCR (qPCR) assays for the detection of six Leishmania species DNA in Colombia
title_short Analytical performance of Four Polymerase Chain Reaction (PCR) and real time PCR (qPCR) assays for the detection of six Leishmania species DNA in Colombia
title_full Analytical performance of Four Polymerase Chain Reaction (PCR) and real time PCR (qPCR) assays for the detection of six Leishmania species DNA in Colombia
title_fullStr Analytical performance of Four Polymerase Chain Reaction (PCR) and real time PCR (qPCR) assays for the detection of six Leishmania species DNA in Colombia
title_full_unstemmed Analytical performance of Four Polymerase Chain Reaction (PCR) and real time PCR (qPCR) assays for the detection of six Leishmania species DNA in Colombia
title_sort analytical performance of four polymerase chain reaction (pcr) and real time pcr (qpcr) assays for the detection of six leishmania species dna in colombia
publisher Frontiers Media S.A.
publishDate 2017
url https://repository.urosario.edu.co/handle/10336/24094
https://doi.org/10.3389/fmicb.2017.01907
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