Proposal of a validation method for automated nucleic acid extraction and RT-qPCR analysis : an example with Bluetongue virus
Elise VandemeulebrouckeKris De ClercqFrank VandenbusscheYves Van Der Stede
CODA-CERVA-VAR
Department of Virology
Molecular Platform
Molecular platformGeneral goal
� Creation of a Molecular Platform for the ‘high-throughput’ RT-qPCR analysis of former list-A pathogens.
� Automatisation
JANUS Automated Workstation (Perkin Elmer)
2
JANUS Automated Workstation (Perkin Elmer)2 extraction robots1 RT-qPCR robot + 2 LightCyclers480 (Roche)
� ExtractionNucleoSpin 96 Virus Core kit (Macherey-Nagel)Vacuum-based protocol
� Real-time RT- PCRRNA Ultrasense kit (Invitrogen)
One-step protocolMultiplexed : virus, internal and external control
� Creation of a Molecular Platform for the ‘high-throughput’ real-time PCR analysis of former list-A pathogens.
� High-throughputExtraction : 12 plates / day 1116 samples / day
Molecular platformGeneral goal
3
Extraction : 12 plates / day PCR : 12 PCR plates / day
� Pathogens :BTV (Bluetongue Virus)
FMDV (Foot and mouth disease Virus)3D
5UTR
CSFV (Classical Swine Fever Virus)AIV (Avian Influenza Virus)
1116 samples / day
RT-qPCR assayQuality Control
Triplex : Virus / Internal Control / External Control
� Controling the sample
Internal Control : endogeneous mRNA (GAPDH) SAMPLE QUALITY
EXTRACTION
4
External Control : synthetic ssRNA (EC-EXTR)
(added to the sample prior to extracion)
� Controling the assay
Negative extraction Control : H2O
Negative PCR Control : H2O
Positive PCR Control : Synthetic RNA
(IC, EC, BTV, FMD 3D, FMD 5UTR, CSF, AI)
EXTRACTION EFFICIENCY
PCR INHIBITION
PCR REACTION
CONTAMINATION
ValidationIntroduction
� Validation of extraction and RT-qPCR
� BTV spiked blood samples
� Based on OIE manual (chapters 1.1.3 and 1.1.5) , internal procedure
� Parameters to be determined :
5
Linearity & Efficiency
Analytical Sensitivity (Limit of Detection)
Analytical Specificity
Intra- and Interrun repeatability
Position effect
Comparison with manual extraction
Cross-contamination
� Vandemeulebroucke et al. Proposal of a validation method for automated nucleic acid
extraction and RT-qPCR analysis: an example with Bluetongue virus (in revision)
Automated extraction procedure
ValidationLinearity & Efficiency
� 10-fold dilution series of BTV spiked blood (-3.14 to 4.86 log10 TCID50 / ml)
5 repeats/dilutionlinear regression analysis
PCR efficiency E (%) :
6
PCR efficiency E (%) :
100 x (101/slope -1)
Linear range :
-1.14 to 4.86 log10 TCID50 / ml
� 2-fold dilution series of BTV spiked blood25 repeatsprobit analysis
Limit Of Detection (95%)
ValidationAnalytical sensitivity
7
Limit Of Detection (95%)
-1.04 log10 TCID50 / ml
Limit Of Detection (95%)
-2.04 log10 TCID50 / sample
� Genetically and clinically related viruses(based on the Fact Sheets of the Center for Food Security & Public Health, College of Vet. Med., Iowa State Universisty)
ValidationAnalytical specificity
8
All were negative for BTV with BTV RT-qPCR
� 2-fold dilution series of BTV spiked blood5 repeats/run Intrarun repeatability5 runs Interrun repeatability Total CV (%)
ValidationIntrarun and interrun repeatability
9
� Positive spiked blood sample96 repeats/plate2 plates/runANOVA analysis
� Comparison of :
ValidationPosition effect
10
� Comparison of :plate 1 versus plate 2 (2 plates simultaneously)row-to-row (pipetting accuracy)column-to-column (pipetting accuracy)inner-to-outer plate (vacuum strenght)
� plates : no effectcolumns : no effectrows : no effectinner-to-outer plate : no effect
� correlation manual vs automated set-up :153 spiked samplesPassing Bablok regression analysis
ValidationCorrelation with manual extraction
11
intercept95% CI: -0,64 to 1,78slope95% CI: 0,93 to 1,01
� Procedures interchangeable
� 48 BTV spiked positive samples & 48 negative samplescheckerboard pattern
ValidationCross contamination
Positive samples
12
Negative samples
Acknowledgements
• Frank Vandenbussche, Molecular Platform
• Kris De Clercq, Development of diagnostic tools for epizootic diseases
• Yves Van Der Stede, Coordination Centre for Veterinary
13
• Yves Van Der Stede, Coordination Centre for Veterinary Diagnostics
• Veterinary and Agrochemical Research Centre (VAR)
• Belgian Federal Agency for the Safety of the Food Chain