PCR-based 16S ribosomal DNA detection technique for Clostridium estertheticum causing spoilage in vacuum-packed chill-stored beef

Chill stored vacuum-packaged meat is sometimes spoiled by psychrotrophic or psychrophilic clostridia. Clostridium estertheticum was first isolated fro m vacuum-packed beef from southern Africa, but has recently been found in b eef originating from northern Europe. This organism is difficult to isolate using conventional methods, and two PCR-based methods have been devised fo r use in measures to control the bacterium in the abattoir and to study its ecology. In the first method, primers were designed having a high annealin g temperature of 65 degrees C to increase specificity, producing a PCR prod uct of 567 bp from the 16S rDNA. Two species of Enterobacteriaceae found in meat cross-reacted in this test, and so it was necessary to use a second s tep, digesting the PCR product with two restriction enzymes. Subsequently a further set of primers was designed, producing a PCR product of 641 bp, an d using an annealing temperature of 60 degrees C. The second procedure was more specific and did not require subsequent restriction analysis of the PC R product. The two sets of primers appeared to have similar sensitivity, de tecting 10-100 cells of C. estertheticum in broth, meat or meat purge (drip ). A semiquantitative method is described for estimating numbers of the tar get bacterium. (C) 1999 Elsevier Science B.V. All rights reserved.