A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw milk

We have developed a quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw milk using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). The designed primers permitted the amplification of a 147 bp DNA fragment from a wide selection of bacteria which may grow in milk at refrigeration temperatures. Amplified PCR products generated using a digoxigenin-labelled primer were heat-denatured before being quantified by an enzyme-linked immunosorbent assay (ELISA). A biotinylated probe immobilized onto streptavidin-coated microplates was used to capture the digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbency differences when assaying milk samples containing bacteria in the range 10(3)-10(7) cfu ml-1. The detection threshold for the PCR-ELISA assay developed in this work is 103 cfu ml-1.