Standardization of a SYBR Green Based Real-Time PCR System for Detection and Molecular Quantification of Babesia bovis and B. bigemina in Water Buffaloes (Bubalus bubalis)
| Autor: | Rosangela Zacarias Machado, Rodrigo Giglioti, Dasiel Obregón, M. D. Rabelo, T. A. Néo, P Alfonso, Thalita B. Bilhassi, Márcia Cristina de Sena Oliveira, Belkis Corona |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Veterinary medicine General Veterinary biology Babesia bovis Parasitemia 030108 mycology & parasitology biology.organism_classification medicine.disease Water Buffaloes 03 medical and health sciences Real-time polymerase chain reaction Reference sample Water buffalo medicine Animal Science and Zoology Bubalus Nested polymerase chain reaction |
| Zdroj: | Journal of Buffalo Science. 5:44-52 |
| ISSN: | 1927-520X |
| DOI: | 10.6000/1927-520x.2016.05.02.4 |
| Popis: | Water buffalo (Bubalus bubalis) is a potential reservoir for Babesia bovis and B. bigemina in tropical regions, but the epidemiological evidence of their reservoir competence is limited, especially due to the lack of diagnostic tests capable of detecting and quantifying the low-level parasitemia present in the carrier animals. In this paper we present the standardization process of a SYBR Green based real-time PCR system (qPCR), consisting of two single qPCR assays, for the detection and quantification of B. bovis and/or B. bigemina. Both assays were optimized in similar protocols, including reagent concentrations and thermocycling parameters, so it is possible its use as a multiple qPCR in a single run. Both single assays showed a suitable analytical performance, especially by allowing detection of a greater number of carrier animals when compared with nested PCR assays (nPCR) against a reference panel of 60 DNA samples extracted from blood of both, infected- and non-infected buffaloes. Furthermore, a mathematical algorithm to convert the qPCR outcomes in percent of infected red blood cell was used, and was found that the estimated parasitemia in carrier buffaloes within the reference sample panels were close to those described in carrier cattle. This method could be a useful tool for epidemiological studies on the participation of the bubaline specie in the epidemic process of bovine babesiosis. |
| Databáze: | OpenAIRE |
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