Single-enzyme amplified fragment length polymorphism and its applicability for Salmonella epidemiology.
Amplified fragment length polymorphism (AFLP) is a PCR-based DNA fingerprinting technique whereby restriction fragments may be visualized without prior knowledge of nucleotide sequences. In AFLP analysis, bacterial genomic DNA is digested with a restriction enzyme and ligated to adapter oligonucleotides. A subset of DNA fragments are then amplified using primers which contain adapter-defined sequences. Selective amplification is achieved by the use of primers containing adapter-defined sequences with one additional arbitrary nucleotide. We used four primers complementary to the adapter sequence, but each differing in the final 3' base that extended into the fragment DNA. The usefulness of these primers for fingerprinting Salmonella enterica was assessed in a hierarchical manner. Using a single-enzyme approach (SAFLP) we have used this method to fingerprint 30 strains of S. enterica, belonging to 14 different serotypes. SAFLP profiles derived from Hind III fragments differentiated between the serotypes. In addition, SAFLP profiles for each serotype differentiated between the phage types and individual strains. The technique is significantly faster to perform than other DNA-based methods and has given reproducible and discriminatory results. This hierarchical SAFLP technique may provide a valuable addition to existing methods for the DNA fingerprinting of S. enterica for epidemiological studies.
Peters
T M
TM
Laboratory of Enteric Pathogens, Central Public Health Laboratory, London, UK. tpeters@phls.org.uk
Threlfall
E J
EJ
D016172:DNA Fingerprinting / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000145:classification* / Q000235:genetics