title: The use of PCR-DGGE to determine bacterial fingerprints for poultry and red meat abattoir effluent.
abstract: Strict legislation and chemical composition monitoring of effluent may be useful, but the data generated do not allow for source tracking, and enforcing legislation remains problematic in the South African setting. These difficulties emphasize the necessity for effluent source traceability. Denaturing gradient gel electrophoresis (DGGE) targeting the V3 region of the 16S rRNA gene was considered as fingerprinting technique for effluent originating from abattoirs slaughtering different animal species. The influence of treatment to remove excess fat from effluent prior to molecular analyses and different PCR approaches on the detection of bacterial diversity were considered. Use of a treatment option to remove fat and a nested PCR approach resulted in up to 51% difference in inter-sample diversity similarity. A robust approach with no pre-treatment to remove PCR inhibitors, such as fat, and direct amplification from genomic DNA yielded optimal/maximal bacterial diversity fingerprints. Repeatable fingerprints were obtained for poultry abattoir effluent over a 4-month period, but profiles for the red meat abattoir varied with maximum similarity detected only 33·2%. Genetic material from faecal indicators Aeromona spp and Clostridium spp were detected. Genera unique to each effluent were present; Anoxybacillus, Patulibacter and Oleispira in poultry abattoir effluent and Porphyromonas and Peptostreptococcus in red meat abattoir effluent.
  
  
  CONCLUSIONS
  This study was the first to demonstrate the application of denaturing gradient gel electrophoresis (DGGE) to construct bacterial diversity fingerprints for high-throughput abattoir effluents. Proved redundancy of fat removal as PCR inhibitor and change in diversity similarity introduced by nested PCR approach. The importance of limiting excessive handling/processing which could lead to misrepresented diversity profiles was emphasized.
authors.affiliation: Department of Life Sciences, Centre for Applied Food Safety and Biotechnology, Central University of Technology, Free State, Bloemfontein, South Africa.
authors.forename: O
authors.initials: O
authors.lastname: de Smidt
mesh_terms: D000003:Abattoirs; D000333:Aeromonas / Q000145:classification / Q000235:genetics; D000818:Animals; D058677:Anoxybacillus / Q000145:classification / Q000235:genetics; D001483:Base Sequence; D003013:Clostridium / Q000145:classification / Q000235:genetics; D016172:DNA Fingerprinting / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D058645:Denaturing Gradient Gel Electrophoresis / Q000379:methods*; D005243:Feces / Q000382:microbiology; D008969:Molecular Sequence Data; D010462:Peptostreptococcus / Q000145:classification / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D017861:Porphyromonas / Q000145:classification / Q000235:genetics; D011200:Poultry / Q000382:microbiology*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D000069466:Red Meat / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D013019:South Africa
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: Green Drop; abattoir effluent; denaturing gradient gel electrophoresis; molecular fingerprint; source tracking; wastewater