A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs

dc.creatorRampazzo, Rita de Cássia Pontello
dc.creatorSolcà, Manuela da Silva
dc.creatorSantos, Liliane Celestino Sales
dc.creatorPereira, Lais de Novaes
dc.creatorGuedes, José Carlos Oliveira
dc.creatorVeras, Patrícia Sampaio Tavares
dc.creatorFraga, Deborah Bittencourt Mothé
dc.creatorKrieger, Marco Aurélio
dc.creatorCosta, Alexandre Dias Tavares
dc.date2017-10-18T18:00:30Z
dc.date2017-10-18T18:00:30Z
dc.date2017
dc.date.accessioned2023-09-26T23:38:46Z
dc.date.available2023-09-26T23:38:46Z
dc.identifierRAMPAZZO, 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.
dc.identifier0304-4017
dc.identifierhttps://www.arca.fiocruz.br/handle/icict/22872
dc.identifier10.1016/j.vetpar.2017.09.009
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8892735
dc.descriptionFINEP (#01.13.0283.00 to M.A.K).
dc.descriptionCanine 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.
dc.formatapplication/pdf
dc.languageeng
dc.publisherElsevier
dc.rightsopen access
dc.subjectLeishmaniose visceral canina
dc.subjectDiagnóstico
dc.subjectGelified qPCR
dc.subjectDuplex qPCR
dc.subjectCanine visceral leishmaniasis
dc.subjectDiagnosis
dc.subjectReady-to-use diagnosis technique
dc.subjectDuplex qPCR
dc.subjectGelified qPCR
dc.titleA ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs
dc.typeArticle


Este ítem pertenece a la siguiente institución