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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20

Steyn, HC, Pretorius, A, McCrindle, CME 10 April 2008 (has links)
Heartwater is a tick borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by Ehrlichia ruminantium and transmitted by the tick Amblyomma hebraeum. The protocols currently used to detect heartwater take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCRTaqMan probe assay to detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E. ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A., Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E., Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A., 2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number. PNAS 102, 838–843]. The pCS20 quantitative real-time PCRTaqMan probe was compared to the currently used pCS20 PCR and PCR/32P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCRTaqMan probe was the most sensitive assay detecting seven copies of DNA/ml of cell culture. All three assays, however, cross react with Ehrlichia canis and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile reaction. The PCR/32P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus, because this new quantitative pCS20 real-time PCRTaqMan probe assay was the most sensitive and can be performed within 2 h it is an effective assay for epidemiological surveillance and monitoring of infected animals.

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