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Ehrlichia ruminantium detection using efficient high-throughput molecular method. [0088]

Cangi N., Pinarello V., Bournez L., Lefrançois T., Albina E., Neves L., Vachiéry N.. 2017. In : Book of abstracts of 9th International conference on tick and tick borne pathogens: One health. Cairns : TTP9-APRC1, p. 56-56. International conference on tick and tick borne pathogens: One health. 9, 2017-08-27/2017-09-01, Cairns (Australie).

In order to improve sample screening capacity and E. ruminantium molecular diagnostics, an automated DNA extraction method for Amblyomma ticks and a new qPCR targeting the pCS20 gene region were successfully developed. A comparison between the new pCS20 Sol1 qPCR (both Sybergreen and Taqman chemistries (TqM)), a previously published pCS20 CowTqM qPCR and the pCS20 nested PCR was carried out. pCS20 Sol1TqM qPCR was found to be as specific as the nested PCR with limited sample contamination and significant gain of time. Concerning the specificity, it did not detect Rickettsia, Anaplasma and Babesia species nor Panola Mountain Ehrlichia, E. chaffeensis and E. canis. In parallel, a tick 16SSG rDNA qPCR was developed for DNA extraction control, showing a good reproducibility of the automatic extraction. The whole method, including the automated DNA extraction and pCS20 Sol1TqM qPCR, demonstrated to be sensitive, specific and highly reproducible with the same limit of detection as the manual DNA extraction and nested PCR. Finally, it allows for 96 samples to be tested in one day compared to four days for manual DNA extraction and nested PCR. The development of a new automated DNA extraction using a DNA/RNA viral extraction kit and qPCR enhances the accuracy of E. ruminantium epidemiological studies, as well as allowing for the improvement of diagnostic capabilities and enhanced turn-over time for heartwater surveillance. In addition, the new method opens new opportunities for large-scale screening of other bacteria and viruses in ticks as well as tick genetic characterization and co-evolution studies.(Texte intégral)

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