Quantitative PCR for Anaplasma Detection

A duplex qPCR assay with minor modifications for the LightCycler real-time machine (Roche Diagnostics) targeting the msp1β gene of A. marginale and the groEL gene of A. centrale, was used to detect Anaplasma spp. in genomic DNA samples as previously described [18 (link)]. DNA extracted from the A. centrale vaccine strain (Onderstepoort Biological Products, Pretoria, South Africa) or field sample 9410 (confirmed to be infected with A. centrale by amplification and sequence analysis of the groEL, msp2 and 16S rRNA genes [18 (link)]) were used as positive controls. Field samples C14 or C57 (obtained from cattle in the Mnisi Community area) were used as positive controls for A. marginale, and molecular grade water as a negative control. To determine A. centrale loads, DNA was extracted from 10-fold serial dilutions of vaccine prepared in uninfected bovine blood. The data were analysed using LightCycler Software version 4.0. (Roche Diagnostics). The linear range of detection and assay efficiency of selected variants were determined as previously described [18 (link)].

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Hove P., Chaisi M.E., Brayton K.A., Ganesan H., Catanese H.N., Mtshali M.S., Mutshembele A.M., Oosthuizen M.C, & Collins N.E. (2018). Co-infections with multiple genotypes of Anaplasma marginale in cattle indicate pathogen diversity. Parasites & Vectors, 11, 5.

Publication 2018

LightCycler LightCycler Software version 4.0

16s rrna Anaplasma Assay Biological products Blood Bovine Diagnostics Dilutions Genes Genomic Rrna genes Sequence analysis Strain Vaccine

Corresponding Organization :

Other organizations : University of Pretoria, Washington State University, National Zoological Gardens of South Africa, Scientific Services

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Variable analysis

independent variables

Modifications to the duplex qPCR assay

dependent variables

Detection of Anaplasma spp. in genomic DNA samples
Anaplasma centrale loads

control variables

LightCycler real-time machine (Roche Diagnostics)
Targeting the msp1β gene of A. marginale and the groEL gene of A. centrale

controls

Positive controls: DNA extracted from the A. centrale vaccine strain (Onderstepoort Biological Products, Pretoria, South Africa) or field sample 9410 (confirmed to be infected with A. centrale by amplification and sequence analysis of the groEL, msp2 and 16S rRNA genes [18 (link)]), field samples C14 or C57 (obtained from cattle in the Mnisi Community area) for A. marginale
Negative control: Molecular grade water

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