High-throughput molecular testing of ticks using a liquid-handling robot.
To meet the need for high-throughput sample testing, DNA extraction kits based on the 96-well plate format have been developed for use with blood and tissue samples. These methods have not been applied to DNA extractions from ticks. To meet this need, we developed a high-throughput method for DNA extraction and polymerase chain reaction (PCR) testing of tick samples. A liquid-handling robot was used to extract DNA in a 96-well binding column plate with vacuum manifold. The quantity, purity, and quality of DNA recovered from Ixodes scapularis Say, 1821 nymphs with this method were reproducible and comparable with existing manual DNA extraction techniques. The DNA yield from pools of five nymphal ticks averaged 0.432 +/- 0.04 microg (95% CI). The robot also prepared real-time PCR reactions in 96-well plates, directly from the extracted DNA. A modification of the existing P20 tool resulted in accurate pipetting of 1- to 2-microl volumes with a reproducibility of +/- 0.038 microl when dispensing 1.0 microl. By using this process, 96 samples can be extracted and tested while reducing human labor to approximately 30 min.
Moriarity
John R
JR
Viral and Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Loftis
Amanda D
AD
Dasch
Gregory A
GA
D000818:Animals; D001096:Arachnid Vectors / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D018884:Ixodes / Q000235:genetics*; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012284:Rickettsia rickettsii / Q000235:genetics / Q000302:isolation & purification; D012371:Robotics / Q000295:instrumentation* / Q000379:methods*
D004247:DNA