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Automated Extraction for
RNA/DNA Combined with
TaqMan Technology and
Cobas Ampliscreen
Elisa Moretti
Process and Analytical Development
KEDRION S.p.A
SoGAT XVI
Paul Ehrlich Institut - Langen, Germany
3rd July 2003
NAT Development
Single extraction for RNA & DNA
Virus Kit
(BIOROBOT 9604 QIAGEN)
AMPLIFICATION & DETECTION
HIV-1, HBV & HCV
KIT Cobas Ampliscreen
(Cobas Amplicor)
ROCHE
PVB19 & HAV
In-house Protocol
(ABI PRISM 7900HT)
APPLERA
Final Results
Preliminary Results
• Instruments validation
according to GMPs
• HCV
12 UI/ml = 100 %
• HAV:
Detection Limit:
33,68 UI/ml
95% C.I.
25,86 - 49,86 UI/ml
HAV-RNA ISS 0201
WHO IS 96/790 NIBSC
• HIV-1
100 UI/ml = 100%
WHO IS 97/656 NIBSC
• HBV
6 UI/ml = 100 %
WHO IS 97/746 NIBSC
Parvovirus B19
• Detection Limit:
133,35 UI/ml
95% C.I.
107,63 - 185,77
• Linearity:
105-103 UI/ml
• Cross-contamination:
Problems with high
PVB19 titre
WHO IS 99/800 NIBSC
• Alternative extraction
QIAamp MinElute Virus
Spin Kit or Ampliscreen
• Amplification & Detection
ABIPrism 7900HT
Evaluation in progress
• Validation:
Quantitative Method
(PA/PH/OMCL (03) 38 2R;
June 2003)
Acknowledgements
• Laboratory of Immunology ISS
Giuliano Gentili
• German Red Cross, Frankfurt
Willi Kurt Roth
• Kedrion
Giampaolo Nardi