Comparison of Four Template Preparation Methods and Optimization of BOX A1R PCR for DNA Fingerprinting of Escherichia coli Isolates

The cycling parameters for BOX A1R PCR were modified and the primer-specific annealing temperature was optimized. Using Trout Lake Escherichia coli isolates, four different template preparation methods were compared. The cell washing, heat lysis, and DNA isolation methods all gave clear and bright band patterns with good reproducibility. The cell washing template preparation method for direct use in PCR seems the most promising, as the procedure was simple and the level of detail obtained in band patterns was comparable to that of the DNA isolation method. It was found that the growth stage of the cells did not affect the outcome, and thus showed that the DNA fingerprinting method was robust in this aspect. The alkaline lysis method appeared to be a poor template preparation method; the method had poor reproducibility and poor output signal. The study that investigated the effect of added NaOH to PCR reactions suggested that NaOH was inhibitory to PCR.