Proportional distribution of the red complex and its individual pathogens after sample storage using the checkerboard DNA-DNA hybridization technique

Abstract

Background: Information on the impact of sample storage prior to analysis by DNA methods is limited. Aims: To investigate the effect of microbial sample storage on bacterial detection and proportional distribution of the red complex and its individual pathogens. Material and Methods: Subgingival plaque samples were analysed by (1) immediate processing, (2) after storage at +4°C for 6 weeks, (3) after storage at -20°C for 6 months or (4) after storage at -20°C for 12 months using the checkerboard DNA-DNA hybridization. Results: Proportional distribution of the red complex did not differ between the first three protocols. However, the total bacterial DNA for pathogens studied decreased significantly in protocols 3 and 4. Relative amounts of Tannerella forsythensis, Porphyromonas gingivalis and Treponema denticola remained stable in the second protocols and changed in an unpredictable way if stored for 6 or 12 months. Conclusions: Results from samples stored for maximum 6 months at -20°C with high proportional amounts of the red complex and T. denticola may be used as an indicator of persistence. All bacterial samples for DNA extraction should be processed following a standardized storage protocol (i.e. samples stored at +4°C for maximum 6 weeks) in order to get comparable qualitative and quantitative results for total DNA, bacterial complexes and individual pathogens. Most representative results are yielded if processing and hybridization could be performed immediately after sampling. © Blackwell Munksgaard, 2005.