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

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Published inApplied and Environmental Microbiology Vol. 66; no. 2; pp. 844 - 849
Main Authors SABAT, G, ROSE, P, HICKEY, W. J, HARKIN, J. M
Format Journal Article
LanguageEnglish
Published Washington, DC American Society for Microbiology 01.02.2000
Subjects
Bacteria
Bacteriological methods and techniques used in bacteriology
Bacteriology
Biological and medical sciences
Blotting, Southern
DNA Primers
Enterobacteriaceae - classification
Enterobacteriaceae - genetics
Enterobacteriaceae - isolation & purification
Escherichia coli
Escherichia coli - classification
Escherichia coli - genetics
Escherichia coli - isolation & purification
Fundamental and applied biological sciences. Psychology
Gene Amplification
Genes
Genes, rRNA - genetics
Methods
Microbiology
Polymerase Chain Reaction - methods
Ribonucleic acid
RNA
RNA, Ribosomal, 16S - genetics
rRNA 16S gene
Sensitivity and Specificity
Soil Microbiology
Soils
Molecular hybridization
Escherichia coli
Ribosomal DNA
Primer
Selectivity
Method
Amplification
Polymerase chain reaction
Soils
Sensitivity
16S-RNA
Bacteria
Detection
Enterobacteriaceae
Online AccessGet full text

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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 degree C, which is 10 degree 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 sub(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.
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 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.
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.
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.
ABSTRACT 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 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.
Author G. Sabat
P. Rose
W. J. Hickey
J. M. Harkin
AuthorAffiliation Department of Bacteriology, 1 Department of Soil Science, 3 and Environmental Toxicology Center, University of Wisconsin—Madison, Madison, Wisconsin 53706-1299 2
AuthorAffiliation_xml – name: Department of Bacteriology, 1 Department of Soil Science, 3 and Environmental Toxicology Center, University of Wisconsin—Madison, Madison, Wisconsin 53706-1299 2
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  fullname: SABAT, G
  organization: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706-1299, United States
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  surname: ROSE
  fullname: ROSE, P
  organization: Environmental Toxicology Center, University of Wisconsin-Madison, Madison, Wisconsin 53706-1299, United States
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  givenname: W. J
  surname: HICKEY
  fullname: HICKEY, W. J
  organization: Environmental Toxicology Center, University of Wisconsin-Madison, Madison, Wisconsin 53706-1299, United States
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  surname: HARKIN
  fullname: HARKIN, J. M
  organization: Environmental Toxicology Center, University of Wisconsin-Madison, Madison, Wisconsin 53706-1299, United States
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Issue 2
Keywords Molecular hybridization
Escherichia coli
Ribosomal DNA
Primer
Selectivity
Method
Amplification
Polymerase chain reaction
Soils
Sensitivity
16S-RNA
Bacteria
Detection
Enterobacteriaceae
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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@facstaff.wisc.edu.
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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...
ABSTRACT 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...
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StartPage 844
SubjectTerms Bacteria
Bacteriological methods and techniques used in bacteriology
Bacteriology
Biological and medical sciences
Blotting, Southern
DNA Primers
Enterobacteriaceae - classification
Enterobacteriaceae - genetics
Enterobacteriaceae - isolation & purification
Escherichia coli
Escherichia coli - classification
Escherichia coli - genetics
Escherichia coli - isolation & purification
Fundamental and applied biological sciences. Psychology
Gene Amplification
Genes
Genes, rRNA - genetics
Methods
Microbiology
Polymerase Chain Reaction - methods
Ribonucleic acid
RNA
RNA, Ribosomal, 16S - genetics
rRNA 16S gene
Sensitivity and Specificity
Soil Microbiology
Soils
Title Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil
URI http://aem.asm.org/content/66/2/844.abstract
https://www.ncbi.nlm.nih.gov/pubmed/10653763
https://www.proquest.com/docview/205950048
https://search.proquest.com/docview/17490094
https://pubmed.ncbi.nlm.nih.gov/PMC91908
Volume 66
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