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Applied and Environmental Microbiology, May 2009, p. 2677-2683, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.02166-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Empirical Testing of 16S rRNA Gene PCR Primer Pairs Reveals Variance in Target Specificity and Efficacy Not Suggested by In Silico Analysis{triangledown}

Sergio E. Morales1 and William E. Holben1,2*

Microbial Ecology Program, Division of Biological Sciences,1 Montana—Ecology of Infectious Diseases Program, The University of Montana, Missoula, Montana2

Received 18 September 2008/ Accepted 20 February 2009

Phylogenetic and "fingerprinting" analyses of the 16S rRNA genes of prokaryotes have been a mainstay of microbial ecology during the last two decades. However, many methods and results from studies that rely on the 16S rRNA gene for detection and quantification of specific microbial taxa have seemingly received only cursory or even no validation. To directly examine the efficacy and specificity of 16S rRNA gene-based primers for phylum-, class-, and operational taxonomic unit-specific target amplification in quantitative PCR, we created a collection of primers based solely on an extensive soil bacterial 16S rRNA gene clone library containing ~5,000 sequences from a single soil sample (i.e., a closed site-specific library was used to create PCR primers for use at this site). These primers were initially tested in silico prior to empirical testing by PCR amplification of known target sequences and of controls based on disparate phylogenetic groups. Although all primers were highly specific according to the in silico analysis, the empirical analyses clearly exhibited a high degree of nonspecificity for many of the phyla or classes, while other primers proved to be highly specific. These findings suggest that significant care must be taken when interpreting studies whose results were obtained with target specific primers that were not adequately validated, especially where population densities or dynamics have been inferred from the data. Further, we suggest that the reliability of quantification of specific target abundance using 16S rRNA-based quantitative PCR is case specific and must be determined through rigorous empirical testing rather than solely in silico.

* Corresponding author. Mailing address: Microbial Ecology Program, Division of Biological Sciences, The University of Montana, Missoula, MT 59812-1006. Phone: (406) 243-6163. Fax: (406) 243-4184. E-mail: bill.holben{at}mso.umt.edu

{triangledown} Published ahead of print on 27 February 2009.

Applied and Environmental Microbiology, May 2009, p. 2677-2683, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.02166-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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