A PCR-BASED METHOD OF DETECTION AND DIFFERENTIATION OF K88-COLI( ADHESIVE ESCHERICHIA)

The objective of this study was to develop a polymerase chain reaction (PCR)-based method to detect and differentiate among Escherichia coli strains containing genes for the expression of 3 antigenic variants o f the fimbrial adhesin K88 (K88ab, K88ac, and K88ad). Five primers wer e designed that allowed detection of K88(+) E. coli, regardless of ant igenic variant, and the separate detection of the ab, ac, and ad varia nts. Primers AM005 and AM006 are 21 base pair (bp) oligomers that corr espond to a region of the K88 operon that is common to all 3 antigenic variants, Primers MF007, MF008, and MF009 are 24-bp oligomers that ma tched variable regions specific to ab, nc, and nti, respectively. Usin g primers AM005 and AM006, a PCR product was obtained that corresponds to a 764-6p region within the large structural subunit of The K88 ope ron common to all 3 antigenic variants. Primer AM005 used with MF007, MF008, or MF009 produced PCR products approximately 500-bp in length f rom within the large structural subunit of the K88 operon of the 3 res pective antigenic variants. Fragments were identified by rates of migr ation on a 1% agarose gel relative to each other as well as to BstEII- digested lambda fragments. This PCR-based method was comparable to the enzyme-linked immunosorbent assay and western blot test in ?he abilit y to differentiate between the antigenic variants. K88(+) E. coli were differentiated from among laboratory strains and detected in ileal sa mples taken from cannulated pigs challenged with a known K88(+) varian t. 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. Detect ion and differentiation of K88(+) E. coli using general and specific p rimers was successful, PCR methods of detection should permit identifi cation of K88(+) antigenic variants regardless of the level of express ion of the antigen.