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Title: PHYLUM AND CLASS SPECIFIC PCR PRIMERS FOR GENERAL MICROBIAL COMMUNITY ANALYSIS

Author
item Blackwood, Christopher
item OAKS, ADAM - UNIV. OF MARYLAND
item Buyer, Jeffrey

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/6/2005
Publication Date: 10/1/2005
Citation: Blackwood, C.B., Oaks, A., Buyer, J.S. 2005. Phylum and class specific PCR primers for general microbial community analysis. Applied and Environmental Microbiology. 71:6193-6198.

Interpretive Summary: Molecular genetic methods are used to monitor microbial populations in soils because the soil community is highly complex and most organisms are unable to grow outside their natural habitat. Most molecular methods are based on the polymerase chain reaction (PCR), which generates a large number of copies of a DNA sequence. These methods use 'universal' PCR primers, which are supposed to amplify DNA from all microorganisms in a particular kingdom. However, these primers are not truly universal, so large numbers of organisms are missed. Also, if differences between samples are detected it is generally not possible to attribute these differences to particular microbial groups. We developed and tested several phylum- and class-specific PCR primers. Because these primers are more specific than those typically used, analyses are more sensitive to changes in populations and communities and results can be related to organismal taxonomy. PCR product generated from soil community DNA using these primers was shown to be derived from the correct groups. These primers may be useful in risk assessment and other studies where it is necessary to determine the impact of a particular treatment, such as introduction of a genetically modified organism or conversion of a farm from conventional to sustainable agriculture, on soil microbial communities.

Technical Abstract: Amplification of a particular DNA fragment from a mixture of organisms by polymerase chain reaction (PCR) is a common first step in methods of examining microbial community structure. The use of domain-specific ribosomal PCR primers implies that any major shift in community structure will be identified. However, sensitivity is lost using domain-specific primers because minor taxa are not detected, and there is random convergence of genetic markers in disparate taxa. The purpose of the current study was to begin to develop a primer set which can be used to replace domain-specific primers. Primers specific for the 16S ribosomal sequences of Alphaproteobacteria, Betaproteobacteria, Bacilli, Actinobacteria, and Planctomycetes, and for the internal transcribed spacer region of Basidiomycota, were examined. Chosen primers were tested through 1. comparison to database sequences and 2. cloning and sequencing from soil community DNA. Eighty-five to 100% of the sequences obtained from clone libraries were found to placed with the groups intended as targets, demonstrating the specificity of the primers under field conditions. It will be important to reevaluate primers over time because of the continual growth of sequence databases and revision of microbial taxonomy.