Date of Award
Master of Science
Frank Masincupp, Neal Schrick
The objective of this study was to develop a Polymerase Chain Reaction (PCR)-based method to detect and differentiate between Escherichia coli possessing genes for the expression of three antigenic variants of the fimbrial adhesin K88 (K88ab, K88ac, and K88ad). Five primers were designed that allowed detection of K88 E. coli, regardless of antigenic variant, as well as the separate detection of ab, ac, and ad variants. Primers AM005 and AM006 were 21 base-pair oligomers that corresponded to a region of the K88 operon that is common to all 3 antigenic variants. Primers MF007, MF008, and MF009 were 24 base-pair oligomers that matched variable regions specific to ab, ac, and ad variants, respectively. Using primers AM005 and AM006, a PCR product was obtained that corresponded to a 764 base-pair region within the large structural subunit of the K88 operon common to all 3 antigenic variants. Primer AM005 used with either MF007, MF008, or MF009 produced PCR products approximately 500 base-pairs in length from within the large structural subunit of the K88 operon of the 3 respective antigenic variants. Fragments were identified by rates of migration on a 1% agarose gel relative to each other as well as to fragments derived from BstEII-digested lambda DNA. This PCR-based method compared well with ELISA and Western Blot tests for the ability to differentiate between the antigenic variants. K88 E. coli were differentiated from among laboratory strains and detected in ileal samples taken from cannulated pigs challenged with a known K88 variant. K88 E. coli were also detected from fecal swabs taken from newly weaned pigs, thus confirming that this PCR-based test could provide a convenient clinical assay for the detection of K88 E. coli. Detection and differentiation of K88 E. coli using general and specific primers were successful. PCR methods of detection should permit identification of K88 antigenic variants regardless of the level of expression of the antigen.
Franklin, Mark Ashburn, "A PCR-Based Method of Detection and Differentiation of K88 Escherichia coli. " Master's Thesis, University of Tennessee, 1996.