High-throughput purification of viral RNA based on novel aqueous chemistry for nucleic acid isolation.
BACKGROUND
Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead-based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive.


METHODS
We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 muL plasma in 1 batch.


RESULTS
The detection limit of the method was comparable to the spin-column-based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R(2)) was >0.99 for the viruses evaluated.


CONCLUSIONS
Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.
Hourfar
Michael K
MK
Institute of Transfusion Medicine and Immunohaematology, German Red Cross, Johann Wolfgang Goethe University, Frankfurt, Germany. khourfar@bsdhessen.de
Michelsen
Uwe
U
Schmidt
Michael
M
Berger
Annemarie
A
Seifried
Erhard
E
Roth
W Kurt
WK
D001322:Autoanalysis; D015497:HIV-1 / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008280:Magnetics; D008567:Membranes, Artificial; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D012371:Robotics; D012492:Salts; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide; D014867:Water / Q000737:chemistry
D008567:Membranes, Artificial; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D012492:Salts; D014867:Water; D012822:Silicon Dioxide