AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY TO DETECT PCR PRODUCTS OF THE RFBS GENE FROM SEROGROUP-D SALMONELLAE - A RAPID SCREENING PROTOTYPE

We describe a digoxigenin-based enzyme-linked immunosorbent assay (DIG -ELISA) following a PCR to detect the amplified lipopolysaccharide rfb S gene as a means for rapid screening of serogroup D salmonellae in st ool specimens, For pure bacterial cultures, the sensitivity of the PCR DIG-ELISA was approximately 10 bacteria. In the presence of stool mat erials, the salmonellae were first isolated by an immunomagnetic separ ation technique with an O9-specific monoclonal antibody, MATy-O9, foll owed by PCR and DIG-ELISA, The corresponding sensitivity was about 10 to 100 bacteria, To evaluate the assay performance clinically, 203 sto ol samples from patients with diarrhea were subjected to the routine c ulture techniques and the PCR ELISA method with overnight enrichment, The conventional culture method identified 145 salmonellae (31 serogro up B, 27 serogroup C, 82 serogroup D, and 5 serogroup E isolates) and 58 non-salmonella bacteria, The PCR ELISA method correctly identified all 82 serogroup D salmonellae (A(405) by ELISA, 2.54 +/- 0.73) but wa s negative for the other Salmonella serogroups (A(405), 0.26 +/- 0.08; n = 63) and non-Salmonella isolates (A(405), 0.16 +/- 0.04; n = 58), In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), along with brief enrichment cultivation of th e samples (from 4 to 16 h), Thus, the PCR DIG-ELISA offers a fast, acc urate, semiquantitative means of detecting infectious agents such as s almonellae, and future robotic automation is possible.