PCR Optimization of BOX-AIR PCR for Microbial Source Tracking of Escherichia coli in Waterways

Different PCR parameters and conditions were investigated to identify the optimal conditions for improved reproducibility and intensity of BOX A1R PCR products. Four isolates of Escherichia coli of either gull or duck origins were cultured, washed, and used as whole cell PCR templates. BOX A1R PCR was performed using template from different growth phases, and by varying PCR annealing temperatures, and the presence/absence of bovine serum albumin (BSA). The results showed that the growth phases of the culture template did not impact either reproducibility or intensity of the PCR bands. Different PCR annealing temperature did affect the intensity of different molecular weight bands and the presence of different bands. Generally, high PCR annealing temperature produced more visible low molecular weight bands and low PCR annealing temperature produced high molecular weight bands. The addition of BSA appeared to have a negative effect on the intensity of the bands. Even though from the results optimal PCR conditions could be approximated from the results, there are other parameters and factors in the modified PCR protocol that should be investigated to further improve the PCR results for accurate microbial source tracking analysis.