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 o f bacteria in refrigerated raw milk using primers designed from conser ved regions in the 16S ribosomal RNA gene (rRNA). The designed primers permitted the amplification of a 147 bp DNA fragment from a wide sele ction 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 immun osorbent assay (ELISA). A biotinylated probe immobilized onto streptav idin-coated microplates was used to capture the digoxigenin-labelled f ragments that were detected with a peroxidase anti-digoxigenin conjuga te. Subsequent enzymic conversion of substrate gave distinct absorbenc e differences when assaying milk samples containing bacteria in the ra nge 10(3)-10(7) cfu ml(-1). The detection threshold for the PCR-ELISA assay developed in this work is 10(3) cfu ml(-1).