Evaluation of a Sandwich Gene Probe Assay for Newcastle Disease Virus.
Abstract
A sandwich gene probe assay was evaluated in comparison to a direct gene probe assay. The target sequence used in each of the assays was a 673 bp DNA fragment of the major nucleocapsid protein gene of NDV. In the direct probe assay, the 673 bp DNA fragment was labelled with digoxigenin and hybridized to unlabelled 673 bp target DNA. In the sandwich assay, the target DNA was detected using two probes. The primary probe was unlabelled, recombinant M13mp18 viral DNA containing the 673 bp gene fragment which hybridized to the 673 bp target DNA. The secondary probe was digoxigenin- labelled M13mp18 DNA which hybridized to the M13 sequences within the primary probe. The sandwich assay resulted in detection limits similar to those demonstrated for the direct assay (1O% molecules of purified target DNA) when molar probe concentrations for the two as says were around 20 pM. When molar probe concentrations in the sandwich assay were increased beyond this, sensitivity decreased and background problems due to non-specific binding became evident. Based on these results, the direct assay is the method of choice since the sandwich assay was no more sensitive than the direct assay, required more probe material and required additional time-consuming probe preparation steps.
Document Details
- Document Type
- Technical Report
- Publication Date
- Feb 01, 1996
- Accession Number
- ADA307319
Entities
People
- Darrin Gray
- Douglas E. Bader
Organizations
- Defence Research and Development Canada