Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples

New Zealand Veterinary Journal
D J PulfordAmj McFadden

Abstract

To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. The T. orientalis type-specific and the HRM qPCR assays displ...Continue Reading

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Citations

Sep 29, 2015·New Zealand Veterinary Journal·Amj McFaddenD J Pulford
Oct 7, 2015·New Zealand Veterinary Journal·Paul Bingham
Jan 26, 2016·Preventive Veterinary Medicine·A M J McFaddenP Bingham
Mar 2, 2021·Veterinary Parasitology·K E LawrenceW E Pomroy

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Datasets Mentioned

BETA
D11046.1

Methods Mentioned

BETA
light microscopy
PCR
electrophoresis
genotyping

Software Mentioned

Primer3
Rad CFX96
Excel
BLAST ( Basic Local Alignment Search Tool
CFX manager
Bio
Geneious
CLUSTALW

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