Blog
About

4
views
0
recommends
+1 Recommend
1 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis Translated title: Acurácia da reação em cadeia da polimerase quantitativa em amostras de pele íntegra congelada e parafinada no diagnóstico da leishmaniose visceral canina

      Read this article at

      ScienceOpenPublisher
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          ABSTRACT The purpose of the present work was to evaluate the accuracy of quantitative polymerase chain reaction (qPCR) performed on samples of fresh frozen tissue (FT) and formalin-fixed, paraffin-embedded (FFPE) healthy skin. This is a validation study conducted with samples from 46 dogs from an endemic area in Brazil. After sample collection, DNA extractions were conducted using commercial kits and qPCR was oriented to kinetoplast DNA (kDNA) targets of the Leishmania infantum species. The results obtained for the FFPE samples showed 63.6% sensitivity and 77.1% specificity, whereas those obtained for the FT samples showed 100% and 48.6%, respectively. Poor agreement was observed for the results of the qPCR technique with FT and FFPE samples. Our results suggest freezing as the most suitable conservation method for the formation of sample databases considering DNA recovery

          Translated abstract

          RESUMO O objetivo deste trabalho foi avaliar a acurácia da PCR quantitativa (qPCR) realizada em amostras de pele íntegra congelada (FT) e parafinada (FFPE). Trata-se de um estudo de validação, com amostras provenientes de 46 cães de uma área endêmica no Brasil. Após as coletas de amostras, as extrações de DNA foram realizadas utilizando-se kits comerciais, e a qPCR foi orientada para alvos do kDNA da espécie Leishmania infantum. Os resultados obtidos para as amostras FFPE foram 63,6% de sensibilidade e 77,1% de especificidade; para as amostras FT, 100% e 48,6%, respectivamente. A concordância dos resultados da técnica de qPCR com amostras FT e FFPE foi pobre. Os resultados sugerem que o congelamento é o método mais adequado de conservação para banco de amostras para recuperação de DNA.

          Related collections

          Most cited references 34

          • Record: found
          • Abstract: found
          • Article: not found

          The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.

          Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.
            Bookmark
            • Record: found
            • Abstract: not found
            • Article: not found

            Manual de Vigilância e Controle da Leishmaniose Visceral

             MS Brasil (2006)
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis.

              The aim of the present study is to highlight the advantages of real-time quantitative PCR intended to aid in the diagnosis and monitoring of canine leishmaniosis. Diagnosis of canine leishmaniosis is extremely challenging, especially in endemic areas, due to the diverse and non-specific clinical manifestations, and due to the high seroprevalence rate in sub-clinical dogs. Veterinarian clinicians are usually confronted with cases that are compatible with the disease, and with several diagnostic tests, sometimes with contradictory results. We have developed a new TaqMan assay, targeting the kinetoplast, applied to 44 samples of bone marrow aspirate or peripheral blood. The dynamic range of detection of Leishmania DNA was established in 7 logs and the limit of detection is 0.001 parasites in the PCR reaction. At the time of diagnosis parasitemia ranges from less than 1 to 10(7)parasites/ml. The ability to quantify the parasite burden allowed: (i) to elucidate the status of positive dogs by conventional PCR, although larger studies are necessary to clarify the dividing line between infection and disease, (ii) to estimate the kinetics of the parasite load and the different response to the treatment in a follow-up and (iii) to validate blood as less invasive sample for qPCR. The continuous data provided by real-time qPCR could solve the dilemma for the clinician managing cases of canine leishmaniosis by differentiating between Leishmania-infected dogs or dogs with active disease of leishmaniosis.
                Bookmark

                Author and article information

                Contributors
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Journal
                abmvz
                Arquivo Brasileiro de Medicina Veterinária e Zootecnia
                Arq. Bras. Med. Vet. Zootec.
                Universidade Federal de Minas Gerais, Escola de Veterinária (Belo Horizonte, MG, Brazil )
                1678-4162
                November 2017
                : 69
                : 6
                : 1443-1450
                Affiliations
                Curitiba orgnameFundação Oswaldo Cruz Brazil
                Rio de Janeiro orgnameFundação Oswaldo Cruz Brazil
                Salvador orgnameFundação Oswaldo Cruz Brazil
                Article
                S0102-09352017000601443
                10.1590/1678-4162-9053

                This work is licensed under a Creative Commons Attribution 4.0 International License.

                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 34, Pages: 8
                Product
                Product Information: SciELO Brazil

                Comments

                Comment on this article