Abstract
BackgroundMolecular 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.ResultsTwo 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.ConclusionsThe 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.
Highlights
Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis
Informed consent was obtained from the dogs’ owners and the person included in the study, and all procedures were approved by the Research Ethics Committee (CEPFIOCRUZ/PE, 42/2010) and by the Ethics Committee for Animal Use (CEUA- FIOCRUZ/RJ, LW-41/10 and LW-1/11) of our institution
Amplicons from pUC18 DNA resulted in bands of 368 (P1) and 316 (P2) base pairs, being suitable for multiplex polymerase chain reaction (PCR) with G3PD (567 bp) and kDNA1 (138 bp)
Summary
Molecular biological methods have become increasingly relevant to the diagnosis and control of infectious diseases, such as leishmaniasis. The quality of the DNA samples extracted from blood and biopsies was evaluated by including a primer system to detect the G3PD gene in two standardized PCR assays for L. infantum (mVL) or L. braziliensis (mACL). The expression of this gene in all mammalian cells ensures its detection in samples whose conditions are suitable for diagnostic tests [13,14,15]. Some known positive samples, with quality assured by the G3PD detection, were PCR-negative for the main DNA target (L. infantum) These results indicated the necessity of ensuring the high quality of each individual sample, and assessing possible losses of minimal amounts of the target parasite’s DNA
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