DEVELOPMENT OF A MICROPLATE GENE PROBE ASSAY OF NEWCASTLE DISEASE VIRUS

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Authors
  1. Bader, D.E.
  2. Lewis, J.
Corporate Authors
Defence Research Establishment Suffield, Ralston ALTA (CAN)
Abstract
A microplate gene probe assay for Newcastle disease virus was developed to take advantage of enhancements in automation and machine-readable quantitation over membrane-based assay formats. A 673 base pair double-stranded DNA fragment of the major nucleocapsid protein gene of Newcastle disease was labeled with a non-radioactive molecule (digoxigenin) and used as a probe against denatured, unlabeled DNA bound to 96-well, polystyrene microtiter plates. The probe/target complex was detected with an anti-digoxigenin antibody conjugated to horseradish peroxidase, followed by colorimetric detection at 405 nm. Technical details in the development and optimization of the microplate assay are presented. The optimized assay had a lower detection limit of about 2x10^7 molecules of DNA under low stringency conditions (Tm-32 degrees Celsius). A membrane-based limit of 10^5-10^6 molecules under high stringency conditions (Tm-1.4 degrees Celsius) indicating that the microplate assay was not as sensitive. Even though the microplate assay was found to be less sensitive relative to the membrane assay, other characteristics such as enhanced capability for automation (plate washing and reading), machine-readable quantitation (simpler, relatively lower cost) and ease of handling and manipulation were realized, favouring the microplate format as an option for fieldable identification systems such as those sent to the Gulf War during Operation Desert Storm.
Report Number
DRES-M-1470 — Memorandum
Date of publication
01 Oct 1995
Number of Pages
38
DSTKIM No
96-02387
CANDIS No
498221
Format(s):
Document Image stored on Optical Disk;Hardcopy

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