RAPID DETECTION OF SALMONELLA SUBSPECIES-I BY PCR COMBINED WITH NONRADIOACTIVE HYBRIDIZATION USING COVALENTLY IMMOBILIZED OLIGONUCLEOTIDE ON A MICROPLATE

A polymerase chain reaction (PCR)-based test was developed for the det ection of Salmonella. One pair of oligonucleotide primers was designed to amplify a 93-bp fragment of a gene required for the invasion of He La cells by Salmonella ser Typhi strain Ty2. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plat es using two oligonucleotides. The capture oligonucleotide was covalen tly linked onto aminated wells of microtiter plates. The detection oli gonucleotide was labelled with biotine. The hybrid molecules were dete cted by avidine conjugated with alkaline phosphatase and chromogenic s ubstrate. The described combination of microplate sandwich hybridisati on and PCR seems to be a suitable method for rapid detection of Salmon ella subspecies I. It only requires a thermal cycler and a conventiona l microtiter reader, and can be readily done on a large scale.