Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis
Background : Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a...
Published in: | Journal of Venomous Animals and Toxins including Tropical Diseases |
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ftdoajarticles:oai:doaj.org/article:8d46b50f21ce420cab9ce27689685c5f 2023-05-15T15:12:58+02:00 Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis Suênia da Cunha Gonçalves-de-Albuquerque Rômulo Pessoa e Silva Rayana Carla Silva de Morais Lays Adrianne Mendonça Trajano-Silva Carlos Gustavo Régis-da-Silva Sinval Pinto Brandão Filho Milena de Paiva-Cavalcanti 2014-05-01T00:00:00Z https://doi.org/10.1186/1678-9199-20-16 https://doaj.org/article/8d46b50f21ce420cab9ce27689685c5f EN eng SciELO http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992014000200326&lng=en&tlng=en https://doaj.org/toc/1678-9199 1678-9199 doi:10.1186/1678-9199-20-16 https://doaj.org/article/8d46b50f21ce420cab9ce27689685c5f Journal of Venomous Animals and Toxins including Tropical Diseases, Vol 20, Iss 0 (2014) Extraction control Multiplex PCR pUC18 Leishmaniasis Diagnosis False-negative result Arctic medicine. Tropical medicine RC955-962 Toxicology. Poisons RA1190-1270 Zoology QL1-991 article 2014 ftdoajarticles https://doi.org/10.1186/1678-9199-20-16 2022-12-31T00:08:41Z Background : Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite’s DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite’s DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.Results : Two primer pairs were designed to detect the plasmid pUC18 and a triplex PCR assay targeting theLeishmania braziliensiskinetoplast DNA, the external control and the internal control was standardized. The triplex PCR assay was assessed for its ability to detect the three target DNA fragments simultaneously. PCR products from pUC18 DNA resulted in bands of 368 (P1) and 316 (P2) base pairs (bp). The triplex PCR optimized with the chosen external control system (P1) allowed the simultaneous detection of the internal control (G3PD – 567 bp) as well as of small quantities (10 pg) of the target parasite’s DNA, detected by amplification of a 138 bp product.Conclusions : The new tool standardized herein enables a more reliable interpretation of PCR results, mainly by contributing to quality assurance of leishmaniasis diagnosis. Furthermore, after simple standardization steps, this protocol could be applied to the diagnosis of other infectious diseases in reference laboratories. This triplex PCR enables the assessment of small losses during the DNA extraction process, problems concerning DNA degradation (sample quality) and the detection ofL. braziliensiskDNA. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Journal of Venomous Animals and Toxins including Tropical Diseases 20 1 16 |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
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English |
topic |
Extraction control Multiplex PCR pUC18 Leishmaniasis Diagnosis False-negative result Arctic medicine. Tropical medicine RC955-962 Toxicology. Poisons RA1190-1270 Zoology QL1-991 |
spellingShingle |
Extraction control Multiplex PCR pUC18 Leishmaniasis Diagnosis False-negative result Arctic medicine. Tropical medicine RC955-962 Toxicology. Poisons RA1190-1270 Zoology QL1-991 Suênia da Cunha Gonçalves-de-Albuquerque Rômulo Pessoa e Silva Rayana Carla Silva de Morais Lays Adrianne Mendonça Trajano-Silva Carlos Gustavo Régis-da-Silva Sinval Pinto Brandão Filho Milena de Paiva-Cavalcanti Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis |
topic_facet |
Extraction control Multiplex PCR pUC18 Leishmaniasis Diagnosis False-negative result Arctic medicine. Tropical medicine RC955-962 Toxicology. Poisons RA1190-1270 Zoology QL1-991 |
description |
Background : Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. Since various factors may affect the sensitivity of PCR assays, including DNA yield and purity, an optimal extraction method is pivotal. Losses of a parasite’s DNA during extraction may significantly impair its detection by PCR and lead to false-negative results. This study proposes a triplex PCR assay targeting the parasite’s DNA, an external control (pUC18) and an internal control (G3PD) for accurate diagnosis of leishmaniasis.Results : Two primer pairs were designed to detect the plasmid pUC18 and a triplex PCR assay targeting theLeishmania braziliensiskinetoplast DNA, the external control and the internal control was standardized. The triplex PCR assay was assessed for its ability to detect the three target DNA fragments simultaneously. PCR products from pUC18 DNA resulted in bands of 368 (P1) and 316 (P2) base pairs (bp). The triplex PCR optimized with the chosen external control system (P1) allowed the simultaneous detection of the internal control (G3PD – 567 bp) as well as of small quantities (10 pg) of the target parasite’s DNA, detected by amplification of a 138 bp product.Conclusions : The new tool standardized herein enables a more reliable interpretation of PCR results, mainly by contributing to quality assurance of leishmaniasis diagnosis. Furthermore, after simple standardization steps, this protocol could be applied to the diagnosis of other infectious diseases in reference laboratories. This triplex PCR enables the assessment of small losses during the DNA extraction process, problems concerning DNA degradation (sample quality) and the detection ofL. braziliensiskDNA. |
format |
Article in Journal/Newspaper |
author |
Suênia da Cunha Gonçalves-de-Albuquerque Rômulo Pessoa e Silva Rayana Carla Silva de Morais Lays Adrianne Mendonça Trajano-Silva Carlos Gustavo Régis-da-Silva Sinval Pinto Brandão Filho Milena de Paiva-Cavalcanti |
author_facet |
Suênia da Cunha Gonçalves-de-Albuquerque Rômulo Pessoa e Silva Rayana Carla Silva de Morais Lays Adrianne Mendonça Trajano-Silva Carlos Gustavo Régis-da-Silva Sinval Pinto Brandão Filho Milena de Paiva-Cavalcanti |
author_sort |
Suênia da Cunha Gonçalves-de-Albuquerque |
title |
Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis |
title_short |
Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis |
title_full |
Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis |
title_fullStr |
Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis |
title_full_unstemmed |
Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis |
title_sort |
tracking false-negative results in molecular diagnosis: proposal of a triplex-pcr based method for leishmaniasis diagnosis |
publisher |
SciELO |
publishDate |
2014 |
url |
https://doi.org/10.1186/1678-9199-20-16 https://doaj.org/article/8d46b50f21ce420cab9ce27689685c5f |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
Journal of Venomous Animals and Toxins including Tropical Diseases, Vol 20, Iss 0 (2014) |
op_relation |
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1678-91992014000200326&lng=en&tlng=en https://doaj.org/toc/1678-9199 1678-9199 doi:10.1186/1678-9199-20-16 https://doaj.org/article/8d46b50f21ce420cab9ce27689685c5f |
op_doi |
https://doi.org/10.1186/1678-9199-20-16 |
container_title |
Journal of Venomous Animals and Toxins including Tropical Diseases |
container_volume |
20 |
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1 |
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16 |
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1766343580283043840 |