Cargando…

Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle

BACKGROUND: The tick-borne apicomplexan bovine parasite Theileria annulata is endemic in many tropical and temperate areas, including Minorca (Balearic Islands, Spain). Real-time PCR is widely used for the detection of piroplasms but quantification is not commonly considered. RESULTS: We developed a...

Descripción completa

Detalles Bibliográficos
Autores principales: Ros-García, Amaia, Nicolás, Antoni, García-Pérez, Ana L, Juste, Ramón A, Hurtado, Ana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3432606/
https://www.ncbi.nlm.nih.gov/pubmed/22889141
http://dx.doi.org/10.1186/1756-3305-5-171
_version_ 1782242232321966080
author Ros-García, Amaia
Nicolás, Antoni
García-Pérez, Ana L
Juste, Ramón A
Hurtado, Ana
author_facet Ros-García, Amaia
Nicolás, Antoni
García-Pérez, Ana L
Juste, Ramón A
Hurtado, Ana
author_sort Ros-García, Amaia
collection PubMed
description BACKGROUND: The tick-borne apicomplexan bovine parasite Theileria annulata is endemic in many tropical and temperate areas, including Minorca (Balearic Islands, Spain). Real-time PCR is widely used for the detection of piroplasms but quantification is not commonly considered. RESULTS: We developed a real-time quantitative PCR (qPCR) assay for the detection and quantification of T. annulata that included an internal amplification control (IAC) to monitor for the presence of potential inhibitors. Specificity, sensitivity, precision, linear range and PCR efficiency were calculated and different methods for transformation of quantification cycle (Cq) values into quantities (Q) were evaluated. The assay was able to detect (100% probability) and quantify (linear response) 100 gene copies, and clinical sensitivity was set at 10 T. annulata per μl of blood. The assay was then validated on 141 bovine blood samples analyzed in parallel by a Luminex® suspension array, showing the utility of the qPCR assay developed here for the detection and quantification of the parasite in field conditions. Once validated it was used to monitor T. annulata parasitaemia throughout a year in 8 carrier animals from a farm in Minorca. CONCLUSIONS: The developed qPCR assay offers a reliable and simple way to quantify T. annulata infection loads, which could prove crucial in studying the role of carrier animals as a source of the infection, or assessing the efficacy of treatment and control measures.
format Online
Article
Text
id pubmed-3432606
institution National Center for Biotechnology Information
language English
publishDate 2012
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-34326062012-09-04 Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle Ros-García, Amaia Nicolás, Antoni García-Pérez, Ana L Juste, Ramón A Hurtado, Ana Parasit Vectors Research BACKGROUND: The tick-borne apicomplexan bovine parasite Theileria annulata is endemic in many tropical and temperate areas, including Minorca (Balearic Islands, Spain). Real-time PCR is widely used for the detection of piroplasms but quantification is not commonly considered. RESULTS: We developed a real-time quantitative PCR (qPCR) assay for the detection and quantification of T. annulata that included an internal amplification control (IAC) to monitor for the presence of potential inhibitors. Specificity, sensitivity, precision, linear range and PCR efficiency were calculated and different methods for transformation of quantification cycle (Cq) values into quantities (Q) were evaluated. The assay was able to detect (100% probability) and quantify (linear response) 100 gene copies, and clinical sensitivity was set at 10 T. annulata per μl of blood. The assay was then validated on 141 bovine blood samples analyzed in parallel by a Luminex® suspension array, showing the utility of the qPCR assay developed here for the detection and quantification of the parasite in field conditions. Once validated it was used to monitor T. annulata parasitaemia throughout a year in 8 carrier animals from a farm in Minorca. CONCLUSIONS: The developed qPCR assay offers a reliable and simple way to quantify T. annulata infection loads, which could prove crucial in studying the role of carrier animals as a source of the infection, or assessing the efficacy of treatment and control measures. BioMed Central 2012-08-13 /pmc/articles/PMC3432606/ /pubmed/22889141 http://dx.doi.org/10.1186/1756-3305-5-171 Text en Copyright ©2012 Ros-García et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Ros-García, Amaia
Nicolás, Antoni
García-Pérez, Ana L
Juste, Ramón A
Hurtado, Ana
Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle
title Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle
title_full Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle
title_fullStr Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle
title_full_unstemmed Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle
title_short Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle
title_sort development and evaluation of a real-time pcr assay for the quantitative detection of theileria annulata in cattle
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3432606/
https://www.ncbi.nlm.nih.gov/pubmed/22889141
http://dx.doi.org/10.1186/1756-3305-5-171
work_keys_str_mv AT rosgarciaamaia developmentandevaluationofarealtimepcrassayforthequantitativedetectionoftheileriaannulataincattle
AT nicolasantoni developmentandevaluationofarealtimepcrassayforthequantitativedetectionoftheileriaannulataincattle
AT garciaperezanal developmentandevaluationofarealtimepcrassayforthequantitativedetectionoftheileriaannulataincattle
AT justeramona developmentandevaluationofarealtimepcrassayforthequantitativedetectionoftheileriaannulataincattle
AT hurtadoana developmentandevaluationofarealtimepcrassayforthequantitativedetectionoftheileriaannulataincattle