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A pool of five oligonucleotides has been used to detect the pathogenic organism Mycobacterium avium subspecies paratuberculosis in PCR-amplified DNA from ruminants. The oligonucleotides were labelled at the 5'-end with three dinitrophenyl reporter groups and hybridised to the target DNA, which was fixed to a nylon membrane by ultraviolet irradiation. Colourimetric detection of the PCR product was carried out using an anti-DNP antibody conjugated to horseradish peroxidase or to alkaline phosphatase. Detection with alkaline phosphatase was more sensitive than with horseradish peroxidase but, in both cases, the PCR product could be easily detected. The DNP labelling system offers an economic and effective alternative to biotin, digoxigenin or fluorescein for the detection of PCR-amplified DNA.

Type

Journal article

Journal

Biomed Pept Proteins Nucleic Acids

Publication Date

1994

Volume

1

Pages

17 - 20

Keywords

Bacterial Typing Techniques, DNA, Bacterial, Dinitrophenols, Molecular Probe Techniques, Mycobacterium avium, Mycobacterium avium subsp. paratuberculosis, Oligonucleotides, Polymerase Chain Reaction