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

Abstract

Chill stored vacuum-packaged meat is sometimes spoiled by psychrotrophic or psychrophilic clostridia. Clostridium estertheticum was first isolated from vacuum-packed beef from southern Africa, but has recently been found in beef originating from northern Europe. This organism is difficult to isolate using conventional methods, and two PCR-based methods have been devised for 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 annealing temperature of 65°C to increase specificity, producing a PCR product 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 step, digesting the PCR product with two restriction enzymes. Subsequently a further set of primers was designed, producing a PCR product of 641 bp, and using an annealing temperature of 60°C. The second procedure was more specific and did not require subsequent restriction analysis of the PCR product. The two sets of primers appeared to have similar sensitivity, detecting 10–100 cells of C. estertheticum in broth, meat or meat purge (drip). A semiquantitative method is described for estimating numbers of the target bacterium.