Comparison of methods for quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples

Two PCR primer sets were developed for the detection and quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for...

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Bibliographic Details
Published in:Applied and environmental microbiology 2000-04, Vol.66 (4), p.1564-1571
Main Authors: Michotey, V, Méjean, V, Bonin, P
Format: Article
Language:English
Subjects:
Bacteria - enzymology
Bacteria - growth & development
Bacteria - isolation & purification
Bacteriological Techniques
Colony Count, Microbial
Cytochromes - genetics
Cytochromes - metabolism
DNA Primers
DNA, Bacterial - analysis
Nitrite Reductases - genetics
Nitrite Reductases - metabolism
Polymerase Chain Reaction - methods
Seawater - microbiology
Sensitivity and Specificity
Water Microbiology
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Summary:Two PCR primer sets were developed for the detection and quantification of cytochrome cd(1)-denitrifying bacteria in environmental marine samples. The specificity and sensitivity of these primers were tested. Both primer sets were suitable for detection, but only one set, cd3F-cd4R, was suitable for the quantification and enumeration of the functional community using most-probable-number PCR and competitive PCR techniques. Quantification of cytochrome cd(1) denitrifiers taken from marine sediment and water samples was achieved using two different molecular techniques which target the nirS gene, and the results were compared to those obtained by using the classical cultivation method. Enumerations using both molecular techniques yielded similar results in seawater and sediment samples. However, both molecular techniques showed 1,000 or 10 times more cytochrome cd(1) denitrifiers in the sediment or water samples, respectively, than were found by use of the conventional cultivation method for counting.
ISSN:0099-2240
DOI:10.1128/AEM.66.4.1564-1571.2000