Update on LAMP Project. OFFLU Technical Meeting, 4-5 April 2012 Royal Holloway, UK Sasan Fereidouni, Anja Globig
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1 Update on LAMP Project OFFLU Technical Meeting, 4-5 April 2012 Royal Holloway, UK Sasan Fereidouni, Anja Globig
2 Early and Rapid Diagnosis of Influenza Need for (rapid) tests with the following properties: basic, but of high quality easy handling and interpretation (under BSL-1) independant of sophisticated equipment safe feasible, affordable robust (storage) availability: easy to procure validated (sensitive, specific) Minimum chance for crosscontamination Possibe to use in developing countries Friedrich-Loeffler-Institut
3 LAMP (Loop mediated Isothermal AMPlification) One version of polymerase amplification methods Rapid molecular method Set of six primers recognizing eight distinct regions on the target gene Result within one hour Isothermal conditions (waterbath / heat block) Single tube
4
5 UV Lamp Normal Light Visual Turbidity Agarose gel Analysis
6 LAMP developed in 2000 (Notomi et al.(2000) Nucleic Acids Res 28: E63) Comparative sensitivities of the LAMP assay and PCR for detection of ALV-J by agarose gel electrophoresis Zhang, X. et al J. Clin. Microbiol. 48(6):
7 LAMP available for (among others): FMD (Dukes et al. (2006): Arch Virol) ND (Pham et al. (2005) J Clin Microbiol) PRRS (Li et al. (2009) J Virol Methods) ASF (Heather et al., (2010) J Virol Methods) AIV H5 (Imai et al. (2006); Eiken, Loopamp ) AIV H7 (Eiken, Loopamp ) AIV H9 (Chen et al. (2008): J Virol Methods) AIV M (Yoshida et al., 2011; J. Vet Med Science) Influenza H1N1 (Kubo et al.(2010): J Clin Microbiol)
8 Evaluation of EIKEN Loopamp (H5/H7) RT-H5/H7 LAMP (Eiken) was on average fold less sensitive than conventional real-time RT-PCR Increasing of sensitivity caused decreasing of specificity
9 Optimization of H5-LAMP IAEA: Bsm DNA polymerase instead of Bst DNA polymerase Modified reaction mix Fluorescence dye EVA-Green Lyophilized all inclusive Master-mix tubes Thermostable: 4 C for months Price (~3 US$)
10 Materials & Methods A panel of LP and HP H5 influenza viruses A panel of all other non-h5 subtypes RNA extraction using QIAamp Viral RNA kit (Qiagen) or rapid-short processing of samples A modified TaqMan one step RTqPCR to compare the results with LAMP. Animal experimet to collect fresh H5-positive diagnostic samples
11 Analytical Sensitivity of IAEA H5 kit
12 Analytical Specificity of IAEA H5 kit Our cut-off IAEA cut-off Positive samples Negative samples & negative Controls
13 Analytical specificity of H5 specific LAMP assay Isolate subtype LAMP 40 min 60 min H5-rRT-PCR Generic (NP) rrt-pcr A/ egyptian goose/germany/r668/10 H1 N1 neg neg neg pos A/swine/Germany/ R158/11 H1 N1 neg pos neg pos A/wild duck/germany/r3111/07 H2 N9 neg neg neg pos A/whooper swan/r281/09 H2 N3 neg pos neg pos A/mallard/Iran/V10/04 H3 N8 neg pos neg pos A/mallard/Germay/R1102/10 H4 N6 neg neg neg pos A/ostrich/Germany/R48/10 H6 N2 neg pos neg pos A/mute swan/germany/r2927/07 H6 N8 neg neg neg pos A/mallard/Iran/V31/04 H7 N3 neg pos neg pos A/anser spec./germany/r44/2006 H8 N3 neg neg neg pos A/mallard/Iran/V16/04 H8 N4 neg neg neg pos A/chicken/Saudi-Arabia/R61/02 H9 N2 neg neg neg pos A/garganey/Iran/K8/03 H9 N2 neg neg neg pos A/eurasian coot/germany/411/10 H10 N8 neg neg neg pos A/pigeon/Israel/543/2008/R713/09 H10 N7 neg neg neg pos A/dk/Kfar/Baruch Israel/2001_R715/09 H11 N2 neg neg neg pos A/duck/Alberta/60/76 H12 N5 neg neg neg pos A/black headed gull/germany/r2622/06 H13N8 neg neg neg pos A/black headed gull/atyrau/2915/08 H13N6 neg pos neg pos A/mallard/Gurjev/263/82 H14 N5 neg neg neg pos A/herring gull/germany/r3309/07 H16N3 neg pos neg pos A/gadwall/Atyrau/2215/07 H16N3 neg pos neg pos
14 Analytical Sensitivity of IAEA H5 kit
15 Analytical Sensitivity of IAEA H5 kit
16 Analytical Sensitivity of IAEA H5 kit H5 Isolate H5-RT-LAMP (last positive dilution) RT-qPCR (last positive dilution) 40 min 60 min dilution Cq A/duck/Potsdam/1402-6/86 (H5N2) A/mallard/Germany/R2557/06 (H5N3) A/ostrich/Germany/R5-10/06 (H5N3) A/mallard/Germany/R409/05 (H5N3) A/Whooper swan/germany/r535/09 (H5N2) A/teal/Germany/WV632/05 (H5N1) A/mallard/Germany/R771/08 (H5N3) A/Mallard/Alberta329/2006 (H5N9) neg
17 Summary of results for IAEA LAMP: H5 LAMP H5 Tetracore H5 LFD PanAI LFD Sensitivity Specificity (40 vs. 60) ++++/ Easy interpretation Cool storage Dependency to instruments Fast results < 1 hour Cost Risk of cross-contmination
18 Advantages Quick results (within maximum 1,5 hours) High sensitivity (more sensitive than rapid test kits, LFD) No expensive equipment needed (theoretically: waterbath) Result can be read by naked eye (fluorescence, turbidity, colour change) Disadvantages Borderline specificity High danger of lab contamination (need for good disposal system)
19 Acknowledgements OIE: Keith Hamilton Bernard Vallat Steve Edwards FAO/OFFLU: Gwenaelle Dauphin IAEA: Hermann Unger FLI: Elke Starick Timm Harder Christian Grund Alex Postel
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Received 31 July 2009/Returned for modification 18 September 2009/Accepted 31 December 2009
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