A comparison of DNA extraction protocols from blood spotted on FTA cards for the detection of tick-borne pathogens by Reverse Line Blot hybridization.
An essential step in the molecular detection of tick-borne pathogens (TBPs) in blood is the extraction of DNA. When cooled storage of blood under field conditions prior to DNA extraction in a dedicated laboratory is not possible, the storage of blood on filter paper forms a promising alternative. We evaluated six DNA extraction methods from blood spotted on FTA Classic®cards (FTA cards), to determine the optimal protocol for the subsequent molecular detection of TBPs by PCR and the Reverse Line Blot hybridization assay (RLB). Ten-fold serial dilutions of bovine blood infected with Babesia bovis, Theileria mutans, Anaplasma marginale or Ehrlichia ruminantium were made by dilution with uninfected blood and spotted on FTA cards. Subsequently, DNA was extracted from FTA cards using six different DNA extraction protocols. DNA was also isolated from whole blood dilutions using a commercial kit. PCR/RLB results showed that washing of 3mm discs punched from FTA cards with FTA purification reagent followed by DNA extraction using Chelex®resin was the most sensitive procedure. The detection limit could be improved when more discs were used as starting material for the DNA extraction, whereby the use of sixteen 3mm discs proved to be most practical. The presented best practice method for the extraction of DNA from blood spotted on FTA cards will facilitate epidemiological studies on TBPs. It may be particularly useful for field studies where a cold chain is absent.
Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany. Electronic address: z.negasi@fu-berlin.de.
Zerihun
Z
Hailemariam
Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany. Electronic address: jabbar.ahmed@gmx.de.
Jabbar Sabir
JS
Ahmed
Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany. Electronic address: peter-henning.clausen@fu-berlin.de.
Peter-Henning
PH
Clausen
Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany. Electronic address: ard.nijhof@fu-berlin.de.
Ard Menzo
AM
Nijhof
D042322:Anaplasma marginale / Q000235:genetics; D000712:Anaplasmosis / Q000097:blood / Q000175:diagnosis / Q000382:microbiology; D000818:Animals; D016793:Babesia bovis / Q000235:genetics; D001404:Babesiosis / Q000097:blood / Q000175:diagnosis / Q000469:parasitology; D002417:Cattle; D002418:Cattle Diseases / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology / Q000469:parasitology; D004269:DNA, Bacterial / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics*; D016995:Ehrlichia ruminantium / Q000235:genetics; D016873:Ehrlichiosis / Q000097:blood / Q000175:diagnosis / Q000662:veterinary; D015151:Immunoblotting / Q000379:methods*; D016794:Theileria / Q000235:genetics; D013801:Theileriasis / Q000097:blood / Q000175:diagnosis / Q000469:parasitology; D017282:Tick-Borne Diseases / Q000097:blood / Q000175:diagnosis / Q000662:veterinary*
D004269:DNA, Bacterial; D016054:DNA, Protozoan
Chelex; DNA extraction; FTA cards; FTA purification reagent; Reverse line blot hybridization; Tick-borne pathogens