Extraction of total nucleic acids from ticks for the detection of bacterial and viral pathogens.
Ticks harbor numerous bacterial, protozoal, and viral pathogens that can cause serious infections in humans and domestic animals. Active surveillance of the tick vector can provide insight into the frequency and distribution of important pathogens in the environment. Nucleic-acid based detection of tick-borne bacterial, protozoan, and viral pathogens requires the extraction of both DNA and RNA (total nucleic acids) from ticks. Traditional methods for nucleic acid extraction are limited to extraction of either DNA or the RNA from a sample. Here we present a simple bead-beating based protocol for extraction of DNA and RNA from a single tick and show detection of Borrelia burgdorferi and Powassan virus from individual, infected Ixodes scapularis ticks. We determined expected yields for total nucleic acids by this protocol for a variety of adult tick species. The method is applicable to a variety of arthropod vectors, including fleas and mosquitoes, and was partially automated on a liquid handling robot.
Crowder
Chris D
CD
Ibis Biosciences, 1896 Rutherford Road, Carlsbad, CA 92008, USA.
Rounds
Megan A
MA
Phillipson
Curtis A
CA
Picuri
John M
JM
Matthews
Heather E
HE
Halverson
Justina
J
Schutzer
Steven E
SE
Ecker
David J
DJ
Eshoo
Mark W
MW
D000818:Animals; D001898:Borrelia / Q000235:genetics / Q000302:isolation & purification; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D016053:RNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017282:Tick-Borne Diseases / Q000235:genetics / Q000517:prevention & control; D013987:Ticks / Q000235:genetics* / Q000382:microbiology* / Q000821:virology*
D017931:DNA Primers; D004269:DNA, Bacterial; D016054:DNA, Protozoan; D004279:DNA, Viral; D012329:RNA, Bacterial; D016053:RNA, Protozoan; D012367:RNA, Viral; D012313:RNA; D004247:DNA