Development of cycling probe technology assay for microfluidic-enabled platform

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Authors
  1. Dickinson-Laing, T.
  2. Pon, J.K.
  3. Mah, M.
  4. Mah, D.
Corporate Authors
Defence Research Establishment Suffield, Ralston ALTA (CAN);Canada West Biosciences, Calgary ALTA (CAN)
Abstract
We have investigated the utility of adapting a cycling probe technology (CPT) assay to a microfluidics format for the identificaiton of the biological warfare simulant Erwinia herbicola. We designed and screened a series of CPT probes for Er. herbiocla, 5'-labelled with either fluorescein or Cy-5 and 3' labelled with biotin, to facilitate detection by capillary electrophoresis with laser-induced fluorescence (CE-LIF). Two of nine probes screened (EH4 and EH8), based on the beta-glucosidase and the tyrosine phenol lyase genes, were found to recognize genomic DNA from Er. herbicola, but were not specific for that organism. EH8 probe was used as a test probe to validate the assay on a benchtop capillary electrophoresis unit, with a final goal to adapt the assay to a microchip format. The lower detection limit of the assay with CE-LIF detection for purified genomic DNA was E5 chromosomal copies. The assay lent itself to adaptation to CE-LIF detection, but with some significant sensitivity and specificity issues. Although absolute quantitation of genomic target was not achieved with this assay, it was able to determine relative estimations of target in pure samples. This assay has not yet been tested with field trial samples. In summary, we have been able to adapt this assay to a rapid CE-LIF detection format suitable for microchip use with a detection time of approximately 5 minutes.
Keywords
Erwinia herbicola;BWA identification;Biological agent simulants;CE-LIF;Capillary Electrophoresis (CE);Genetic analysis;Laser Induced Fluorescence (LIF);Nucelic acid probes;Cycling Probe Technology (CPT);Gene probes
Report Number
DRES-CR-1999-083 — Contract Report
Date of publication
01 Apr 1999
Number of Pages
112
DSTKIM No
CA010089
CANDIS No
514419
Format(s):
Hardcopy;Document Image stored on Optical Disk

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