Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis

dc.creatorAlbuquerque, Suênia da Cunha Gonçalves de
dc.creatorSilva, Rômulo Pessoa e
dc.creatorMorais, Rayana Carla Silva de
dc.creatorSilva, Lays Adrianne Mendonça Trajano
dc.creatorSilva, Carlos Gustavo Régis da
dc.creatorBrandão Filho, Sinval Pinto
dc.creatorCavalcanti, Milena de Paiva
dc.date2017-11-24T13:40:36Z
dc.date2017-11-24T13:40:36Z
dc.date2014
dc.date.accessioned2023-09-27T00:10:46Z
dc.date.available2023-09-27T00:10:46Z
dc.identifierALBUQUERQUE, Suênia da Cunha Gonçalves de et al. Tracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis. Journal of Venomous Animals and Toxins including Tropical Diseases, v. 20, n. 16, p. 1-6, 22 Apr. 2014.
dc.identifier1678-9199
dc.identifierhttps://www.arca.fiocruz.br/handle/icict/23341
dc.identifier10.1186/1678-9199-20-16
dc.identifier1678-9199
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8898235
dc.descriptionCarlos Gustavo Régis da Silva - Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil. Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta a informação no documento.
dc.descriptionBackground: 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 the Leishmania braziliensis kinetoplast 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 of L. braziliensis kDNA.
dc.formatapplication/pdf
dc.languageeng
dc.publisherBMC
dc.rightsopen access
dc.subjectControle de extração
dc.subjectReação em Cadeia da Polimerase Multiplex
dc.subjectpUC18
dc.subjectLeishmaniose
dc.subjectDiagnóstico
dc.subjectResultado falso negativo
dc.subjectExtraction control
dc.subjectMultiplex PCR
dc.subjectpUC18
dc.subjectLeishmaniasis
dc.subjectDiagnosis
dc.subjectFalse-negative result
dc.subjectAnimais
dc.subjectDiagnóstico
dc.subjectReações Falso-Negativas
dc.subjectReação em Cadeia da Polimerase
dc.subjectLeishmaniose
dc.subjectpatologia
dc.titleTracking false-negative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis
dc.typeArticle


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