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Foodborne enterotoxigenic Escherichia coli: Identification and enumeration on nitrocellulose membrane by enzyme immunoassay

1986; Elsevier BV; Volume: 3; Issue: 2 Linguagem: Inglês

10.1016/0168-1605(86)90030-9

1879-3460

D. B. Shah, U.S. Rhea,

Bacterial Genetics and Biotechnology

A solid-phase enzyme immunoassay capable of direct enumeration of Escherichia coli that produce heat-labile enterotoxin (LT) has been developed. Pure or mixed cultures of bacteria or artificially contaminated foods were plated on nitrocellulose membranes placed on semisynthetic agar containing lincomycin. After overnight growth, the colonies were lysed in situ and were reacted with rabbit antiserum to LT. The presence of rabbit IgG in a colony was detected by goat antirabbit IgG peroxidase conjugate. Results of 71 strains tested by this method were in complete accord with LT assays performed by the mouse Y-1 adrenal cells. Three foods, namely, oysters, raw milk and Brie cheese, containing 2 × 106, 1.3 × 105 and 3.9 × 106 total plate count, respectively, were artificially contaminated with known levels of toxigenic E. coli. Upon analysis of membrane filters on which a range of 1 × 103 to 104 cells was deposited, 1 to 10 LT+E. coli could be enumerated with a recovery greater than 85%. The test on foods can be completed in approximately 30 h. The test is sensitive enough to visualize 1 ng of pure LT contained in 1 mm2 area of the membrane.