DEVELOPMENT AND EVALUATION OF A NON-RADIOACTIVE, COLORIMETRIC, MEMBRANE-BASED GENE PROBED ASSAY FOR NEWCASTLE DISEASE VIRUS

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Authors
  1. Bader, D.E.
Corporate Authors
Defence Research Establishment Suffield, Ralston ALTA (CAN)
Abstract
A non-radioactive, membrane-based, colorimetric gene probe assay was developed for Newcastle disease virus (NDV) to evaluate the potential of gene probes as tools for the identification of biological agents. Two gene probes for the NDV major nucleocapsid protein gene were evaluated, namely, (i) a digoxigenin-labelled, double-stranded DNA fragment of approximately 673 base pairs designated as dig-NDVNP673 and (ii) a 5'-fluorescein-labelled, single-stranded oligonucleotide probe of 20 bvases, designated as NDVNP-PB4. Detection limits of E5 -E6 molecules of NDV target DNA were achieved using dig-NDVNP673, however this probe was found to cross-react with bacterial plasmid DNA (pBR328), even under highly stringent assay conditions (TM-1.4C). The oligonucleotide probe, NDVNP-PB4, did not cross-react with pBR328 DNA, even under low stringency conditions (Tm-29C), and generated a detection limit of E7 -E8 molecules for NDV target DNA. However, the oligonucleotide probe cross-reacted with herring sperm DNA and lambda bacteriophage DNA under these conditions. When the stringency was increased during the post-hybridization wash step, from TM-29C to Tm-13C, cross-reactivity was eliminated without compromising assay sensitivity. TRUNCATED
Keywords
Digoxigenin;Gene Probe Assay;Reverse Transcription Polymerase Chain Reaction;Nucleocapsid Protein Gene
Report Number
DRES-617 —
Date of publication
01 Dec 1994
Number of Pages
37
DSTKIM No
95-05080
CANDIS No
153820
Format(s):
Document Image stored on Optical Disk;Hardcopy

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