Proposal of a validation method for automated nucleic acid extraction and RT-qPCR analysis : an example with Bluetongue virus

Transcription

1 Proposal of a validation method for automated nucleic acid extraction and RT-qPCR analysis : an example with Bluetongue virus Elise Vandemeulebroucke Kris De Clercq Frank Vandenbussche Yves Van Der Stede CODA-CERVA-VAR Department of Virology Molecular Platform

2 Molecular platform General goal Creation of a Molecular Platform for the highthroughput RT-qPCR analysis of former list-a pathogens. Automatisation JANUS Automated Workstation (Perkin Elmer) 2 extraction robots 1 RT-qPCR robot + 2 LightCyclers480 (Roche) Extraction NucleoSpin 96 Virus Core kit (Macherey-Nagel) Vacuum-based protocol Real-time RT- PCR RNA Ultrasense kit (Invitrogen) One-step protocol Multiplexed : virus, internal and external control 2

3 Molecular platform General goal Creation of a Molecular Platform for the highthroughput real-time PCR analysis of former list-a pathogens. High-throughput Extraction : 12 plates / day PCR : 12 PCR plates / day 1116 samples / day Pathogens : BTV (Bluetongue Virus) FMDV (Foot and mouth disease Virus) 3D 5UTR CSFV (Classical Swine Fever Virus) AIV (Avian Influenza Virus) 3

4 RT-qPCR assay Quality Control Triplex : Virus / Internal Control / External Control Controling the sample Internal Control : endogeneous mrna (GAPDH) External Control : synthetic ssrna (EC-EXTR) (added to the sample prior to extracion) Controling the assay Negative extraction Control : H 2 O Negative PCR Control : H 2 O Positive PCR Control : Synthetic RNA (IC, EC, BTV, FMD 3D, FMD 5UTR, CSF, AI) SAMPLE QUALITY EXTRACTION EFFICIENCY PCR INHIBITION CONTAMINATION PCR REACTION 4

5 Introduction Validation of extraction and RT-qPCR BTV spiked blood samples Based on OIE manual (chapters and 1.1.5), internal procedure Parameters to be determined : Linearity & Efficiency Analytical Sensitivity (Limit of Detection) Analytical Specificity Intra- and Interrun repeatability Position effect Comparison with manual extraction Cross-contamination Automated extraction procedure Vandemeulebroucke et al. Proposal of a validation method for automated nucleic acid extraction and RT-qPCR analysis: an example with Bluetongue virus (in revision) 5

6 Linearity & Efficiency 10-fold dilution series of BTV spiked blood (-3.14 to 4.86 log 10 TCID 50 / ml) 5 repeats/dilution linear regression analysis PCR efficiency E (%) : 100 x (10 1/slope -1) Linear range : to 4.86 log 10 TCID 50 / ml 6

7 Analytical sensitivity 2-fold dilution series of BTV spiked blood 25 repeats probit analysis Limit Of Detection (95%) log 10 TCID 50 / ml Limit Of Detection (95%) log 10 TCID 50 / sample 7

8 Analytical specificity 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) All were negative for BTV with BTV RT-qPCR 8

9 Intrarun and interrun repeatability 2-fold dilution series of BTV spiked blood 5 repeats/run Intrarun repeatability 5 runs Interrun repeatability Total CV (%) 9

10 Position effect Positive spiked blood sample 96 repeats/plate 2 plates/run ANOVA analysis 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 effect columns : no effect rows : no effect inner-to-outer plate : no effect 10

11 Correlation with manual extraction correlation manual vs automated set-up : 153 spiked samples Passing Bablok regression analysis intercept 95% CI: -0,64 to 1,78 slope 95% CI: 0,93 to 1,01 Procedures interchangeable 11

12 Cross contamination 48 BTV spiked positive samples & 48 negative samples checkerboard pattern Positive samples Negative samples 12

13 Acknowledgements Frank Vandenbussche, Molecular Platform Kris De Clercq, Development of diagnostic tools for epizootic diseases 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 13