Document Detail


Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water.
MedLine Citation:
PMID:  16751534     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2= 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.
Authors:
Alice Layton; Larry McKay; Dan Williams; Victoria Garrett; Randall Gentry; Gary Sayler
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Applied and environmental microbiology     Volume:  72     ISSN:  0099-2240     ISO Abbreviation:  Appl. Environ. Microbiol.     Publication Date:  2006 Jun 
Date Detail:
Created Date:  2006-06-05     Completed Date:  2006-08-30     Revised Date:  2013-06-07    
Medline Journal Info:
Nlm Unique ID:  7605801     Medline TA:  Appl Environ Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4214-24     Citation Subset:  IM    
Affiliation:
The University of Tennessee, Center for Environmental Biotechnology, 676 Dabney Hall, Knoxville, TN 37996-1605, USA. alayton@utk.edu
Data Bank Information
Bank Name/Acc. No.:
GENBANK/AY597127;  AY597128;  AY597129;  AY597130;  AY597131;  AY597132;  AY597133;  AY597134;  AY597135;  AY597136;  AY597137;  AY597138;  AY597139;  AY597140;  AY597141;  AY597142;  AY597143;  AY597144;  AY597145;  AY597146;  AY597147;  AY597148;  AY597149;  AY597150;  AY597151;  AY597152;  AY597153;  AY597154;  AY597155;  AY597156;  AY597157;  AY597158;  AY597159;  AY597160;  AY597161;  AY597162;  AY597163;  AY597164;  AY597165;  AY597166;  AY597167;  AY597168;  AY597169;  AY597170;  AY597171;  AY597172;  AY597173;  AY597174;  AY597175;  AY597176;  AY597177;  AY597178;  AY597179;  AY597180;  AY597181;  AY597182;  AY597183;  AY597184;  AY597185;  AY597186;  AY597187;  AY597188;  AY597189;  AY597190;  AY597191;  AY597192;  AY597193;  AY597194;  AY597195;  AY597196;  AY597197;  AY597198;  AY597199;  AY597200;  AY597201;  AY597202;  AY597203;  AY597204;  AY597205;  AY597206
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MeSH Terms
Descriptor/Qualifier:
Animals
Bacteroides / classification,  genetics*
Base Sequence
Cattle
DNA Primers
DNA, Bacterial / genetics
DNA, Ribosomal / genetics
Feces / microbiology*
Humans
Molecular Sequence Data
Phylogeny
Polymerase Chain Reaction / methods*
RNA, Bacterial / genetics
RNA, Ribosomal, 16S / genetics*
Water Pollution*
Chemical
Reg. No./Substance:
0/DNA Primers; 0/DNA, Bacterial; 0/DNA, Ribosomal; 0/RNA, Bacterial; 0/RNA, Ribosomal, 16S
Comments/Corrections

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