The differentiation of Carnobacterium divergens using the random amplification of polymorphic DNA polymerase chain reaction technique S. Lai 1 , H. Shojaei 2 and L.N. Manchester 1 1 Institute of Biological Sciences, University of Wales, Aberystwyth and 2 Department of Agricultural and Environmental Science, University of Newcastle upon Tyne, Newcastle upon Tyne, UK 0094/00: received 2 February 2000, revised and accepted 22 February 2000 S. LAI, H. SHOJAEI AND L.N. MANCHESTER. 2000. The potential of the randomly ampli®ed polymorphic DNA polymerase chain reaction (RAPD-PCR) technique to differentiate Carnobacterium divergens from other members of the genus Carnobacterium was examined. A numerical analysis of the genomic pro®les obtained demonstrated that it was possible to differentiate the C. divergens strains from other Carnobacterium strains using this technique. The heterogeneity observed in the representatives of the species C. piscicola adds further weight to the suggestion in other taxonomic studies that subspecies of this species exist. INTRODUCTION The genus Carnobacterium contains strains that are recov- ered from a variety of sources ranging from foods such as poultry, meats, cheeses and seafoods (Thornley and Sharpe 1959; Hitchener et al. 1982; Mauguin and Novel 1994; Millie Âre et al. 1994), to isolates from ®sh (Baya et al. 1991; Starliper et al. 1992; Jùburn et al. 1999) and anoxic Antarctic lake waters (Franzmann et al. 1991). The identi®cation of Carnobacterium strains can prove dif®cult, in particular the distinction between the species C. divergens and C. piscicola which are said to be phenotypi- cally similar. This similarity, together with disagreement over the actual phenotypic characters which will distin- guish between them (Holzapfel and Gerber 1983; Shaw and Harding 1985; Collins et al. 1987; Borch and Molin 1988; Montel et al. 1991), has not always made identi®ca- tion of strains straightforward. Over the past few years workers have proposed the use of molecular methods to overcome the dif®culties encountered using the classical phenotypic methods, e.g. protein pro®les (Dicks et al. 1995) and species-speci®c probes (Brooks et al. 1992). PCR-based methods have an advantage over some other molecular techniques as they can produce rapid and reliable results. The randomly ampli®ed polymorphic DNA (RAPD) PCR technique is a modi®cation of the polymerase chain reaction which can be used to produce genomic ®n- gerprints of the strains under examination. The genomic DNA is ampli®ed with single oligonucleotide primers of arbitrary sequence, resulting in the ampli®cation of several discrete DNA products (Welsh and McClelland 1990; Williams et al. 1990). Ampli®cation products are separated on agarose gels and visualized by ethidium bromide stain- ing. This technique has been successfully applied to epide- miological studies and for the examination of variability within bacterial species such as Bacillus thuringiensis subsp israelensis (Ankarloo et al. 2000), Burkholderia cepacia (Okazaki et al. 1999), Listeria spp. (Wagner et al. 1999) and MRSA (Telecco et al. 1999). However, the suitability of this technique for the differentiation of members of the genus Carnobacterium has not been considered. The aim of this study was to investigate the usefulness of RAPD-PCR for the differentiation of 16 representative strains of the genus Carnobacterium, and in particular, its ability to differentiate strains of C. divergens from those of C. piscicola. MATERIALS AND METHODS Cultivation of bacterial strains Sixteen strains (Table 1) were cultivated on brain heart infusion (BHI) agar and incubated at 25  C for 3 d. Three loopfuls of culture were then transferred to 30 ml BHI broth and incubated overnight at 25  C. DNA extraction The guanidium thiocyanate DNA extraction procedure described by Pitcher et al. (1989) was used for the small- Correspondence to: L.N. Manchester, Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DA, UK (e-mail: llm@aber.ac.uk). Letters in Applied Microbiology 2000, 30, 448452 = 2000 The Society for Applied Microbiology