Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach
Abstract INTRODUCTION: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol develo...
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Sociedade Brasileira de Medicina Tropical (SBMT)
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| Series: | Revista da Sociedade Brasileira de Medicina Tropical |
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| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822017002300350&lng=en&tlng=en |
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| author | Lays Adrianne Mendonça Trajano-Silva Rômulo Pessoa-e-Silva Suênia da Cunha Gonçalves-de-Albuquerque Rayana Carla Silva de Morais Cíntia Nascimento da Costa-Oliveira Tayná Correia de Goes Milena de Paiva-Cavalcanti |
| author_facet | Lays Adrianne Mendonça Trajano-Silva Rômulo Pessoa-e-Silva Suênia da Cunha Gonçalves-de-Albuquerque Rayana Carla Silva de Morais Cíntia Nascimento da Costa-Oliveira Tayná Correia de Goes Milena de Paiva-Cavalcanti |
| author_sort | Lays Adrianne Mendonça Trajano-Silva |
| collection | DOAJ |
| description | Abstract INTRODUCTION: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol developed for the simultaneous detection of Leishmania infantum DNA and sample quality control. METHODS: After preliminary tests with the newly designed TaqMan® probes for the two targets ( L. infantum and glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene), the duplex qPCR protocol was optimized. For the evaluation of the standardized protocol, human blood samples were tested (n=68) and the results were compared to those obtained by reference diagnostic techniques. Statistical analyses included percentage agreement and the Kappa ( k ) coefficient. RESULTS: The detection limit of L. infantum DNA reached 2x10 2 fg (corresponding to ~1 parasite) per µL of blood (ε: 93.9%). The percentage agreement obtained between the duplex VL qPCR and the reference techniques was individually obtained as follows: molecular: 88.3% ( k =0.666; 95% CI 0.437-0.894, good), and serological: 81.7% ( k =0.411; 95% CI 0.125-0.697, moderate). Between the reference techniques, the percentage agreement was 86.7% ( k =0.586; 95% CI 0.332-0.840, moderate). CONCLUSIONS: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers. |
| first_indexed | 2024-12-12T19:16:53Z |
| format | Article |
| id | doaj.art-5b029d485da84d7d97cb60778a43fb36 |
| institution | Directory Open Access Journal |
| issn | 1678-9849 |
| language | English |
| last_indexed | 2024-12-12T19:16:53Z |
| publisher | Sociedade Brasileira de Medicina Tropical (SBMT) |
| record_format | Article |
| series | Revista da Sociedade Brasileira de Medicina Tropical |
| spelling | doaj.art-5b029d485da84d7d97cb60778a43fb362022-12-22T00:14:43ZengSociedade Brasileira de Medicina Tropical (SBMT)Revista da Sociedade Brasileira de Medicina Tropical1678-984950335035710.1590/0037-8682-0012-2017S0037-86822017002300350Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approachLays Adrianne Mendonça Trajano-SilvaRômulo Pessoa-e-SilvaSuênia da Cunha Gonçalves-de-AlbuquerqueRayana Carla Silva de MoraisCíntia Nascimento da Costa-OliveiraTayná Correia de GoesMilena de Paiva-CavalcantiAbstract INTRODUCTION: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol developed for the simultaneous detection of Leishmania infantum DNA and sample quality control. METHODS: After preliminary tests with the newly designed TaqMan® probes for the two targets ( L. infantum and glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene), the duplex qPCR protocol was optimized. For the evaluation of the standardized protocol, human blood samples were tested (n=68) and the results were compared to those obtained by reference diagnostic techniques. Statistical analyses included percentage agreement and the Kappa ( k ) coefficient. RESULTS: The detection limit of L. infantum DNA reached 2x10 2 fg (corresponding to ~1 parasite) per µL of blood (ε: 93.9%). The percentage agreement obtained between the duplex VL qPCR and the reference techniques was individually obtained as follows: molecular: 88.3% ( k =0.666; 95% CI 0.437-0.894, good), and serological: 81.7% ( k =0.411; 95% CI 0.125-0.697, moderate). Between the reference techniques, the percentage agreement was 86.7% ( k =0.586; 95% CI 0.332-0.840, moderate). CONCLUSIONS: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822017002300350&lng=en&tlng=enVisceral leishmaniasisDiagnosisHIV/VL co-infectionSample quality controlDuplex qPCR |
| spellingShingle | Lays Adrianne Mendonça Trajano-Silva Rômulo Pessoa-e-Silva Suênia da Cunha Gonçalves-de-Albuquerque Rayana Carla Silva de Morais Cíntia Nascimento da Costa-Oliveira Tayná Correia de Goes Milena de Paiva-Cavalcanti Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach Revista da Sociedade Brasileira de Medicina Tropical Visceral leishmaniasis Diagnosis HIV/VL co-infection Sample quality control Duplex qPCR |
| title | Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
| title_full | Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
| title_fullStr | Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
| title_full_unstemmed | Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
| title_short | Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach |
| title_sort | standardization and evaluation of a duplex real time quantitative pcr for the detection of leishmania infantum dna a sample quality control approach |
| topic | Visceral leishmaniasis Diagnosis HIV/VL co-infection Sample quality control Duplex qPCR |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0037-86822017002300350&lng=en&tlng=en |
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