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)

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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

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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

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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 :

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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 (%) :

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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

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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

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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

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� Positive spiked blood sample96 repeats/plate2 plates/runANOVA analysis

� Comparison of :

ValidationPosition effect

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� 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

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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

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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

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• 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