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United States Department of Agriculture

Agricultural Research Service

Title: BIOTINYLATED PROTEINS OF PASTEURELLA MULTOCIDA AND PASTEURELLA HAEMOLYTICA CAN CAUSE FALSE POSITIVE REACTIONS WITH BIOTINYLATED PROBES IN COLONY LIFT-HYBRIDIZATION ASSAYS

Author
item REGISTER, KAREN

Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 27, 1997
Publication Date: N/A

Interpretive Summary: Swine Respiratory disease is an extremely costly problem for swine producers. Once an outbreak of respiratory disease is recognized, it is necessary to identify the specific microorganism responsible so that appropriate measures can be taken to stop its spread and minimize the severity of disease. The procedures currently used to identify bacteria are often time-consuming and difficult for inexperienced laboratory workers. Recently developed diagnostic tests based upon detection of specific pieces of DNA are more rapid, easy to perform, and highly accurate. However, during the development of a new DNA-based test for identification of bacteria that cause swine respiratory disease, it was discovered that one of the components frequently included in the test caused false positive reactions when certain types of bacteria were present in the test sample. Therefore, this component should not be included in DNA-based tests for swine respiratory disease pathogens. This information will permit the development of more accurate diagnostic tests and research tools for use by scientists, clinical laboratory technicians, and veterinarians.

Technical Abstract: During the development of a hybridization assay for concurrent detection of B. bronchiseptica and toxigenic strains of P. multocida from swine, it became apparent that false positive reactions were obtained with P. multocida whenever a biotinylated probe was used in the procedure. Biotinylated probes did not result in false positive reactions when they were used with genomic DNA on Southern blots. Further investigation revealed that residual biotinylated proteins remaining on the colony lift membrane were binding to the streptavidin conjugate used for development. This caused an apparent positive result in the absence of base-pairing between the probe and the target DNA. Western blots indicated that two major proteins of approximately 38 kDa and 105 kDa, present in both whole cell extracts and extracts enriched in outer membrane proteins, were responsible for the avidin-binding activity. Additional experiments demonstrated the presence of these proteins in several different strains o P. multocida and P. haemolytica, with diverse capsular and somatic serotypes and isolated from a variety of hosts. Although others have described the presence of biotinylated proteins in bacteria that can interfere with Western blotting, this is the first report indicating that such proteins can interfere with a nucleic acid-based assay. Therefore, the use of biotinylated probes specific for P. multocida or P. haemolytica should be avoided in colony lift hybridization assays. Biotinylated probes with other specificities should also be avoided whenever there is a possibility that these two organisms might be present on plates from which the colony lifts are produced.

Last Modified: 8/27/2014
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