Automated extraction of avian influenza virus for rapid detection using real-time RT-PCR.
BACKGROUND
Highly pathogenic H5N1 avian influenza (AI) poses a grave risk to human health. An important aspect of influenza control is rapid diagnosis.


OBJECTIVE
This study describes the efficiency of AI-RNA extraction utilizing silica-based magnetic beads with robotics and its detection with an influenza A matrix gene real-time RT-PCR from tracheal swabs, and compares it to virus isolation and manual spin column extractions.


METHODS
Analytical sensitivity was assessed by performing dilution analysis and detection of H2N2 AI viral RNA. Diagnostic sensitivity and specificity was assessed by analyzing tracheal swabs collected from H7N2 infected and uninfected chickens.


RESULTS
Both manual and robotic extractions detected AI virus at 1log(10)EID(50)/ml. Diagnostic sensitivity and specificity of matrix gene detection with the automated extraction method for chicken tracheal swab specimens was similar to that of virus isolation and the manual extraction method. There were only three discordant results among 212 tested specimens.


CONCLUSIONS
The main advantages of automated robotic viral nucleic acid extraction are high throughput processing; hands-free operation; and reduction in human and technical error. This study demonstrates successful detection of influenza A virus with magnetic beads utilizing the Qiagen MagAttract cell kit on a BioRobot M48 platform.
Tewari
Deepanker
D
Microbiology Services, Pennsylvania Veterinary Laboratory, Harrisburg, PA 17110, USA. dtewari@state.pa.us
Zellers
Corey
C
Acland
Helen
H
Pedersen
Janice C
JC
D000818:Animals; D001331:Automation; D002642:Chick Embryo; D002645:Chickens / Q000821:virology; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis*; D008280:Magnetics; D008863:Microspheres; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000302:isolation & purification; D012371:Robotics; D012680:Sensitivity and Specificity; D014132:Trachea / Q000821:virology
D012367:RNA, Viral