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Open AccessArticle

An Evaluation of Quantitative PCR Assays (TaqMan® and SYBR Green) for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates

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Department of Agriculture & Fisheries, Agri-Science Queensland, Animal Science, Dutton Park QLD 4102, Australia
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Department of Agriculture & Fisheries, Biosecurity Queensland, Tick Fever Centre, Wacol QLD 4076, Australia
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Queensland Alliance for Agriculture & Food Innovation, University of Queensland, Centre for Animal Science, St Lucia QLD 4072, Australia
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Authors to whom correspondence should be addressed.
Academic Editor: Ulrike Munderloh
Vet. Sci. 2016, 3(3), 23; https://doi.org/10.3390/vetsci3030023
Received: 10 June 2016 / Revised: 30 August 2016 / Accepted: 7 September 2016 / Published: 13 September 2016
(This article belongs to the Special Issue Comparative Studies in Tick-Borne Diseases in Animals and Humans)
Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation) showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample. View Full-Text
Keywords: babesiosis; carrier; diagnosis; qPCR; cytochrome b; genotyping; ITS1; vaccines babesiosis; carrier; diagnosis; qPCR; cytochrome b; genotyping; ITS1; vaccines
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Zhang, B.; Sambono, J.L.; Morgan, J.A.T.; Venus, B.; Rolls, P.; Lew-Tabor, A.E. An Evaluation of Quantitative PCR Assays (TaqMan® and SYBR Green) for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates. Vet. Sci. 2016, 3, 23.

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