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Applied and Environmental Microbiology, February 2000, p. 844-849, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil

G. Sabat,1 P. Rose,2 W. J. Hickey,2,3,* and J. M. Harkin2,3

Department of Bacteriology,1 Department of Soil Science,3 and Environmental Toxicology Center, University of Wisconsin---Madison, Madison, Wisconsin 53706-12992

Received 7 September 1999/Accepted 22 November 1999

A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminating E. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72°C, which is 10°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 concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detecting E. coli DNA in heterogeneous DNA samples, such as those extracted from soil.

* Corresponding author. Mailing address: Department of Soil Science, University of Wisconsin---Madison, Madison, WI 53706-1299. Phone: (608) 262-9018. Fax: (608) 265-2595. E-mail: wjhickey{at}facstaff.wisc.edu.

Applied and Environmental Microbiology, February 2000, p. 844-849, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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