A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs
| Autor(a) principal: | |
|---|---|
| Data de Publicação: | 2017 |
| Outros Autores: | , , , , , , , |
| Tipo de documento: | Artigo |
| Idioma: | eng |
| Título da fonte: | Repositório Institucional da FIOCRUZ (ARCA) |
| Texto Completo: | https://www.arca.fiocruz.br/handle/icict/22872 |
Resumo: | FINEP (#01.13.0283.00 to M.A.K). |
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Rampazzo, Rita de Cássia PontelloSolcà, Manuela da SilvaSantos, Liliane Celestino SalesPereira, Lais de NovaesGuedes, José Carlos OliveiraVeras, Patrícia Sampaio TavaresFraga, Deborah Bittencourt MothéKrieger, Marco AurélioCosta, Alexandre Dias Tavares2017-10-18T18:00:30Z2017-10-18T18:00:30Z2017RAMPAZZO, R. C. P. et al. A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs. Veterinary Parasitology, v. 246, p. 100–107, 2017.0304-4017https://www.arca.fiocruz.br/handle/icict/2287210.1016/j.vetpar.2017.09.009FINEP (#01.13.0283.00 to M.A.K).Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, BrasilUniversidade Federal da Bahia. Escola de Medicina Veterinária. Departamento de Medicina Veterinária Preventiva e Produção Animal. Salvador, BA, BrasilUniversidade Federal da Bahia. Escola de Medicina Veterinária. Departamento de Medicina Veterinária Preventiva e Produção Animal. Salvador, BA, BrasilUniversidade Federal da Bahia. Escola de Medicina Veterinária. Departamento de Medicina Veterinária Preventiva e Produção Animal. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, Brasil / Instituto de Ciência e Tecnologia de Doenças Tropicais. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Patologia e Biointervenção. Salvador, BA, Brasil / Universidade Federal da Bahia. Escola de Medicina Veterinária. Departamento de Medicina Veterinária Preventiva e Produção Animal. Salvador, BA, Brasil / Instituto de Ciência e Tecnologia de Doenças Tropicais. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, BrasilFundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, BrasilCanine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precise CVL diagnosis would allow for a faster and more specific treatment. Quantitative PCR (qPCR) is a sensitive and specific technique that can diagnose CVL and also monitor parasite load in the animal during the course of the infection or treatment. The aim of this study was to develop a ready-to-use (gelified and freezer-free) duplex qPCR for the identification of infected animals. We combined a new qPCR protocol that detects the canine 18S rRNA gene with an existing protocol for L. infantum kDNA detection, creating a duplex qPCR. This duplex method was then developed into a ready-to-use format. The performance of the duplex and singleplex reactions were compared in the traditional format (liquid and freezer-stored). Furthermore, the duplex qPCR performance was compared between the ready-to-use and traditional formats. The singleplex and new duplex qPCR exhibited the same detection limit in the traditional format (0.1 parasites/reaction). The ready-to-use format showed a detection limit of 1 parasite/reaction without affecting the reaction efficiency. The performance of the new qPCR protocol in the two formats was assessed using canine tissue samples from 82 dogs in an endemic CVL area that were previously characterized by standard serological and parasitological protocols. Splenic aspirates provided a higher rate of positivity (92.9%) followed by skin (50%) and blood (35.7%). The reported detection limits were observed for all tissues studied. Our results show that the amplification of L. infantum kDNA and canine DNA in a single tube, using either the traditional or ready-to-use format, exhibited the same diagnostic performance as amplification of the parasite kDNA alone. The detection of the host gene strengthens the qPCR results by confirming the presence and quality of DNA in the samples and the absence of polymerase inhibitors. The ready-to-use duplex qPCR format has many advantages. By joining two qPCR protocols into one, more results can be obtained in the same amount of time with reduced costs and embedded quality control. Reagents are preloaded and stored on the plate, reducing the operator's hands-on time to set up a reaction, as well as decreasing manipulation steps, which reduces the risk of mistakes or contamination. Thus, the ready-to-use duplex format turns qPCR into a robust, easy-to-use tool, which could help increase the availability of qPCR for CVL diagnosis.engElsevierLeishmaniose visceral caninaDiagnósticoGelified qPCRDuplex qPCRCanine visceral leishmaniasisDiagnosisReady-to-use diagnosis techniqueDuplex qPCRGelified qPCRA ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogsinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da FIOCRUZ (ARCA)instname:Fundação Oswaldo Cruz (FIOCRUZ)instacron:FIOCRUZLICENSElicense.txtlicense.txttext/plain; charset=utf-82991https://www.arca.fiocruz.br/bitstream/icict/22872/1/license.txt5a560609d32a3863062d77ff32785d58MD51ORIGINALRampazzo RCP A ready-to-use....pdfRampazzo RCP A 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| dc.title.pt_BR.fl_str_mv |
A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs |
| title |
A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs |
| spellingShingle |
A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs Rampazzo, Rita de Cássia Pontello Leishmaniose visceral canina Diagnóstico Gelified qPCR Duplex qPCR Canine visceral leishmaniasis Diagnosis Ready-to-use diagnosis technique Duplex qPCR Gelified qPCR |
| title_short |
A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs |
| title_full |
A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs |
| title_fullStr |
A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs |
| title_full_unstemmed |
A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs |
| title_sort |
A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs |
| author |
Rampazzo, Rita de Cássia Pontello |
| author_facet |
Rampazzo, Rita de Cássia Pontello Solcà, Manuela da Silva Santos, Liliane Celestino Sales Pereira, Lais de Novaes Guedes, José Carlos Oliveira Veras, Patrícia Sampaio Tavares Fraga, Deborah Bittencourt Mothé Krieger, Marco Aurélio Costa, Alexandre Dias Tavares |
| author_role |
author |
| author2 |
Solcà, Manuela da Silva Santos, Liliane Celestino Sales Pereira, Lais de Novaes Guedes, José Carlos Oliveira Veras, Patrícia Sampaio Tavares Fraga, Deborah Bittencourt Mothé Krieger, Marco Aurélio Costa, Alexandre Dias Tavares |
| author2_role |
author author author author author author author author |
| dc.contributor.author.fl_str_mv |
Rampazzo, Rita de Cássia Pontello Solcà, Manuela da Silva Santos, Liliane Celestino Sales Pereira, Lais de Novaes Guedes, José Carlos Oliveira Veras, Patrícia Sampaio Tavares Fraga, Deborah Bittencourt Mothé Krieger, Marco Aurélio Costa, Alexandre Dias Tavares |
| dc.subject.other.pt_BR.fl_str_mv |
Leishmaniose visceral canina Diagnóstico Gelified qPCR Duplex qPCR |
| topic |
Leishmaniose visceral canina Diagnóstico Gelified qPCR Duplex qPCR Canine visceral leishmaniasis Diagnosis Ready-to-use diagnosis technique Duplex qPCR Gelified qPCR |
| dc.subject.en.pt_BR.fl_str_mv |
Canine visceral leishmaniasis Diagnosis Ready-to-use diagnosis technique Duplex qPCR Gelified qPCR |
| description |
FINEP (#01.13.0283.00 to M.A.K). |
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2017 |
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2017-10-18T18:00:30Z |
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2017-10-18T18:00:30Z |
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2017 |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/article |
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article |
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RAMPAZZO, R. C. P. et al. A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs. Veterinary Parasitology, v. 246, p. 100–107, 2017. |
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https://www.arca.fiocruz.br/handle/icict/22872 |
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0304-4017 |
| dc.identifier.doi.none.fl_str_mv |
10.1016/j.vetpar.2017.09.009 |
| identifier_str_mv |
RAMPAZZO, R. C. P. et al. A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs. Veterinary Parasitology, v. 246, p. 100–107, 2017. 0304-4017 10.1016/j.vetpar.2017.09.009 |
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