bioEPS - A quantitative real -time PCR A quantitative real-time PCR … · 2009. 4. 30. · rnc cta...
Transcript of bioEPS - A quantitative real -time PCR A quantitative real-time PCR … · 2009. 4. 30. · rnc cta...
A quantitative real-time PCR
assay for Ehrlichia ruminantium
using pCS20
A quantitative realA quantitative real--time PCR time PCR
assay for Ehrlichia ruminantiumassay for Ehrlichia ruminantium
using pCS20using pCS20
Helena Steyn
Onderstepoort Veterinary Institute,
South Africa
Introduction• Ehrlichia ruminantium is the causative
agent of heartwater
• Obligate intracellular parasite that infects endothelial cells of the host
E. ruminantium colonies in endothelial cells
E. ruminantium organisms
Cell nucleus
pCS20 probe assay
• Fragment contains two overlapping genes:
– rnc and ctaG
Welgevonden genome sequence (Collins 2005) et al.,
rnc ctaG
HH1F HH2Rprobe (AB128-129)
279 bp
Aims of study
• pCS20 real-time PCR TaqMan probe method to detect the presence of E. ruminantium in blood and ticks
• Quantify parasites in cell cultures and experimentally infected sheep and ticks
• Identify and characterise new genotypes of Ehrlichia ruminantium in South Africa
• Development of Heartwater vaccine
Comparison of the sensitivity of three pCS20
assays using Welgevonden infected cell
culture material
Dilution Copy Number
(copies/µl) PCR
PCR/32
P-
probe TaqMan
10-1
7 x 107 + + +
10-2
7 x 106 + + +
10-3
7 x 105 + + +
10-4
7 x 104 + + +
10-5
7 x 103 + + +
10-6
7 x 102 + + +
10-7
7 x 101 - + +
10-8
7 x 100 - - +
Specificity of pCS20 assays
using genomic DNA
Samples PCR 32P-probe TaqMan
Probe
Copy Number
(copies/µl)
Welgevonden + + + 7 x 108
Mara87/7 + + + 1 x 108
Vosloo + + + 3 x 106
Blaauwkrans + + + 1 x 109
Kwanyanga + + + 4 x 108
Ball3 + + + 4 x 106
E. canis + + + 1 x 104
E. chaffeensis + + + 2 x 103
T. parva - - - 3 x 106
A. centrale - - - 4 x 106
Detection of pCS20 in
experimentally infected sheep
Day PCR PCR/32P-
probe
Real-time
PCR
0-10 - - -
11 - 5+/5 -
12 - 5+/5 5+/5
13 5+/5 5+/5 5+/5
14 5+/5 5+/5 5+/5
15 n/d n/d n/d
Parasites determined in
experimentally infected sheep
Sheep number Average parasite copies per ml blood
861 1 x 109
862 2 x 109
869 4 x 108
870 7 x 108
871 4 x108
Conclusions
�The pCS20 real-time PCR was more sensitive than the standard PCR and 32P-probe when applied to cell
culture and tick samples.
�A faster diagnosis can be made within 2 hours instead
of 5 days.
� It is also potentially less expensive to use on a routine basis since it is less time consuming.
�DNA in the samples could be quantified, making it
suitable for diagnostic and epidemiological studies.
Acknowledgements
• ARC-OVI:
– Dr A Pretorius
– Dr M van Kleef
– Dr E Zweygarth
– Mrs A Josemans
– Ms E Faber
• Funding:
– European Union
– Red meat Research and Development Trust, SA
• Other:
– Prof C McCrindle,
University of Pretoria
– Prof C Steinmann,
Tshwane University of Technology,
Pretoria