Selective and sensitive method for PCR amplification of Escherichia coli 16S rRNA genes in soil

A set of PCR primers targeting 16S rRNA gene sequences was designed, and PC R parameters were optimized to develop a robust and reliable protocol for s elective amplification of Escherichia coil 16S rRNA genes. The method was c apable of discriminating E. coil from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coil occurred on ly when the annealing temperature in the PCR was elevated to 72 degrees C, which is 10 degrees C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl2 concentrati on. The PCR protocol developed can be completed in less then 2 h and, by us ing Southern hybridization, has a detection limit of ca. 10 genomic equival ents per reaction. The method was demonstrated to be effective for detectin g E. coil DNA in heterogeneous DNA samples, such as those extracted from so il.