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Padlock- ASMD molecular protocol

WP7: On-Site Confirmation and Monitoring Detection device for padlock-RCA NA amplification products Q-detect meeting , Ljubljana Feb 18, 2013. Padlock- ASMD molecular protocol. Padlock-ASMD technology. Highly stringent identification Isothermal base protocol

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Padlock- ASMD molecular protocol

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  1. WP7: On-SiteConfirmation and MonitoringDetectiondevice for padlock-RCANA amplificationproductsQ-detectmeeting,Ljubljana Feb 18, 2013

  2. Padlock- ASMD molecularprotocol

  3. Padlock-ASMD technology • Highly stringent identification • Isothermal base protocol • Handles relatively ”dirty ”matrices • High-precision quantification • Sensitivity on par with real-time PCR • Intrinsically high multiplexing • Amenable to automation with high throughput in small footprint

  4. Deliverables • Padlock protocols delivered • Probe design input delivered • Prototype device manufactured • Multiple flow channel layout realized • Low-cost lasers evaluated and selected • Low-cost cameras evaluated • Battery operation possible

  5. Detectorprototype • 9-plex capabilityusing 3colors x 3 channelssimultaneously. (Alexa488/Cy3/Cy5) • Replaceable flow channel • PCR striploader • 24V power supply • Movie

  6. Padlock- ASMD molecular protocol

  7. Investigationlow-cost lasers • Comparision of high(Cobolt) and low(CNI) budget laser ar 532 nm wavelength. • Lower efficiency of CNI laser, probably due to imperfect beam shape, but similar overall performance. Significant price advantage. Measured no of RCP Concentration of rolling circle products (RCP)

  8. Detectorsensitivity • Baseline sensitivity of detector • Sensitivity allows precise detection of low levels (tens)of input genomes givenstandard C2CA conditionsin Cy3 and Cy5 channels. Lower sensitivity in Alexa488channel. Measured no of RCP Concentration of RCP (fM)

  9. Multiplexing Detection of eight different probes targeting sequences of eight different bacterial sequences. Four ligation reactions were performed, targeting seven of the target sequences at a constant concentration. One of the target sequences, V harveyii, was titrated in steps of ten. The ligations were amplified by RCA and thereafter split into three hybridization reactions.

  10. Detectorprototype: development potential • Combinatorial staining allows 7 combinations per channel using 3 colors. Using1 combination for reference and one for debris discrimination would give 5*3=15 plex detection • PCR strip loader van be replaced with 96-well sampler • Future possibility: replace reader with array solution GOR X

  11. Q-linea business overview • BioDefence • Q-linea develops technology for identification of bio warfare agents / both nucleic acid and protein-based • Customers from Sweden, Europe and discussions ongoing with US Army • Research • Q-linea continues to take part in several government and EU funded research projects Q-linea has currently 15 employees and plans to hire additional 2-3 persons during 2013 • Diagnostics • Q-linea move into the field of antibiotic resistance diagnostics • Time for sepsis diagnosis & bacterial resistance pattern identification will be reduced from several days to hours (1 Bn € market within EU) • This will dramatically influence treatment & survival of severe septic patients

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