NDLI: Comparative Evaluation of Quantitative Polymerase Chain Reaction Methods for Routine Enumeration of Specific Bacterial DNA in Aquatic Samples

Content Provider	Springer Nature Link
Author	Iue, Daisuke Wada, Katsushi Sei, Kazunari Ike, Michihiko Fujita, Masari
Copyright Year	2005
Abstract	Three quantitative polymerase chain reaction (PCR) methods, the internal standard method (IS-PCR), competitive PCR (cPCR) and most probable number-PCR (MPN-PCR), were compared in terms of their ability to quantify specific bacterial DNA in environmental samples. Serially diluted Pseudomonas putida BH, the target bacterium, was inoculated into sterilized potassium phosphate buffer (PPB), river water and activated sludge, total DNA was extracted, and the number of pheB genes carried by P. putida BH in each sample was enumerated. IS-PCR and cPCR could not quantify the pheB gene at low concentrations (1.0 × 10$^{3}$ copies ml$^{-1}$ in all samples and 1.0 × 10$^{4}$ copies ml$^{--1}$ in some samples) and tended to give overestimations because of differences in amplification efficiencies between pheB gene and the internal standard/competitor in a reaction tube. Although reproducibility of MPN-PCR was slightly lower than that of the other two methods, MPN-PCR was the most sensitive, enabling us to quantify the pheB gene at 1.0 × 10$^{3}$ copies ml$^{--1}$, and it had a good correlation with the inoculum size of P. putida BH. These results suggest that MPN-PCR is the best suited for routine microbial monitoring in natural environmental samples because of the simple handling, the ease of modification as occasion demands and the wide detection range, especially at low cell densities of the target microbe.
Starting Page	1029
Ending Page	1035
Page Count	7
File Format	PDF
ISSN	09593993
Journal	World Journal of Microbiology and Biotechnology
Volume Number	21
Issue Number	6-7
e-ISSN	15730972
Language	English
Publisher	Kluwer Academic Publishers
Publisher Date	2005-01-01
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Competitive polymerase chain reaction environmental samples internal standard method most probable number-polymerase chain reaction quantitative polymerase chain reaction method Biotechnology Applied Microbiology Microbiology Environmental Biotechnology Biochemistry
Content Type	Text
Resource Type	Article
Subject	Physiology Medicine Applied Microbiology and Biotechnology Biotechnology

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