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 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 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 MgCl(2) 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.
Sabat
G
G
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706-1299, USA.
Rose
P
P
Hickey
W J
WJ
Harkin
J M
JM
D015139:Blotting, Southern; D017931:DNA Primers; D004755:Enterobacteriaceae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D005784:Gene Amplification; D020459:Genes, rRNA / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D012988:Soil Microbiology
D017931:DNA Primers; D012336:RNA, Ribosomal, 16S