Phylum- and Class-Specific PCR Primers for General Microbial Community Analysis

Blackwood, Christopher B.; Oaks, Adam W.; Buyer, Jeffrey S.

doi:10.1128/aem.71.10.6193-6198.2005

Cited by 99 publications

(76 citation statements)

References 41 publications

(24 reference statements)

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“…In silico analysis of the group-specific PCR primers Blackwood et al (2005) used ARB to develop five group-specific primers including primers for four of the groups of bacteria investigated in this study. Ashelford et al (2002) compared in detail the PRIMROSE and ARB programs and concluded that in many cases it was possible to identify better oligonucleotide probes (judged by in silico analysis) using PRIMROSE rather than ARB.…”

Section: Resultsmentioning

confidence: 99%

Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities

et al. 2008

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Phylum-and class-specific PCR primers were tested for the production of clone libraries and for denaturing gradient gel electrophoresis (DGGE) analysis of complex bacterial communities. Primers were designed to specifically amplify 16S rRNA gene fragments of the phyla Bacteroidetes, Planctomycetes and Firmicutes, of three classes of the phylum Proteobacteria, the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria, and of the Cyanobacteria (including chloroplast 16S rRNA genes). The specificity of the seven primer pairs was tested by producing clone libraries from environmental DNA samples from mesotrophic (Norwegian coastal) and oligotrophic (Northern Atlantic Gyre) environments. Five of the seven primer pairs specifically amplified target 16S rRNA gene sequences. Exceptions were the Betaproteobacteria-and Firmicutes-specific primers, which were relatively successful with coastal water mesocosm samples but less so with the Northern Atlantic Gyre sample. Phylogenetic analysis of sequences from the Gammaproteobacteria clone library revealed that the coastal sample yielded a number of clones that clustered within clades that belong to the oligotrophic marine Gammaproteobacteria (OMG) group, indicating that this group is not confined exclusively to the oligotrophic environment. Comparison of the bacterial diversity of the environmental DNA sample from the coastal and the open ocean using a two-or three-step nested PCR-DGGE process revealed significant differences in the bacterial communities. The application of the group-specific primers provides a higher resolution genetic fingerprinting approach than existing DGGE primer sets.

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Section: Resultsmentioning

confidence: 99%

Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities

et al. 2008

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“…In the first round of PCR, the primers F984GC (5'gc-AACGCGAAGAACCTTAC 3')/R1492 (5' TACGG(C/T)TA CCTTGTTACGACTT 3'), F243 (5' GGATGAGCCCGCGGCCTA 3')/R1492 and BLS342F (5' CAGCAGTAGGGAATCTTC 3')/R1392 (5' ACGGGCGGTGTGTACA 3') were used to amplify 16S rDNA gene regions specific to Bacteria domain, Actinobacteria and Firmicutes phyla, respectively [28,29]. Primer F984GC contained a GCclamp at the 5'end [28].…”

Section: Amplification and Denaturing Gradient Gel Electrophoresis (Dmentioning

confidence: 99%

Assessment of vaginal microbiota in Brazilian women with and without bacterial vaginosis and comparison with Nugent score

Oliveira¹,

Diniz²,

Fernandes³

et al. 2018

J Infect Dev Ctries

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Introduction: Bacterial vaginosis (BV) is characterized by the depletion of Lactobacillus spp. population and increase of other species, especially Gardnerella vaginalis and Atopobium vaginae. This study aimed to investigate the vaginal microbiota structure of Brazilian women with and without BV according to Nugent Score and to assess the correlation among Nugent score and the quantification of BV-associated bacteria. Methodology: Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) assay was employed to characterize the vaginal microbiota structure. Quantification of Lactobacillus spp., G. vaginalis, A. vaginae, Mobiluncus sp. and M. hominis were determined by quantitative real-time PCR (qPCR). Results: Clustering by PCR-DGGE revealed differences in microbial structure of the different patient groups. Gardnerella vaginalis, A. vaginae, M. hominis and Mobiluncus sp. were detected at high loads in BV-associated microbiota. Quantification of Lactobacillus spp. showed an inverse correlation with Nugent score while the loads of G. vaginalis, A. vaginae, M. hominis and Mobiluncus sp. indicated a direct correlation with this method. Conclusions: Despite Nugent score is considered the gold standard for BV diagnosis, qPCR stands out as a useful tool for bacteria quantification and an alternative for BV diagnosis. Vaginal microbiota is a complex microbial community although there is a common core among BV and non-BV women. Investigation of vaginal microbiota structure may contribute to the development of tools for diagnosis improvement and therapeutic regimen optimization.

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“…Recently, many efforts have been made to develop phylum-or higher taxonomic group-specific PCR primers (Blackwood et al, 2005;Dopheide et al, 2008;Scheckenbach et al, 2010;Stoeck et al, 2009). The main problem encountered in these previous studies is a difficulty in designing higher taxonomic groupspecific primers that discriminate between target groups among all eukaryotes that exist in an environmental sample.…”

Section: Introductionmentioning

confidence: 99%

“…However, predominant organisms or ones possessing genes with sequences preferentially amplified by PCR will be detected before to rare species because the eukaryotic organism communities in most environmental samples are too complex and diverse to be thoroughly analyzed in a single study. For this reason, rare species or small taxonomic groups will not be evenly represented in the sampling data even if mass sequencing methods are used (Blackwood et al, 2005;Nolte et al, 2010;Scheckenbach et al, 2010;Shanks et al, 2011). Rare species or minor taxonomic groups, however, may be more important than quantitatively dominant species when analyzing community structures due to their narrow range of distribution and susceptibility to minute habitat alterations or ecological disturbances (Blackwood et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

“…Recently, high-throughput sequencing methods such as pyrosequencing have been combined with a molecular survey to assess the entire spectrum of eukaryotes belonging to communities in environmental samples from various habitats (Blackwood et al, 2005;Nolte et al, 2010;Scheckenbach et al, 2010;Shanks et al, 2011). Conventional approaches use universal primers specific for an SSU rRNA gene targeting all organisms in an environmental sample.…”

Section: Introductionmentioning

confidence: 99%

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Development of Single-Nucleotide Polymorphism-Based Phylum-Specific PCR Amplification Technique: Application to the Community Analysis Using Ciliates as a Reference Organism

Jung

Kim

Ryu

et al. 2012

Molecules and Cells

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Despite recent advance in mass sequencing technologies such as pyrosequencing, assessment of culture-independent microbial eukaryote community structures using universal primers remains very difficult due to the tremendous richness and complexity of organisms in these communities. Use of a specific PCR marker targeting a particular group would provide enhanced sensitivity and more in-depth evaluation of microbial eukaryote communities compared to what can be achieved with universal primers. We discovered that many phylum-or groupspecific single-nucleotide polymorphisms (SNPs) exist in small subunit ribosomal RNA (SSU rRNA) genes from diverse eukaryote groups. By applying this discovery to a known simple allele-discriminating (SAP) PCR method, we developed a technique that enables the identification of organisms belonging to a specific higher taxonomic group (or phylum) among diverse types of eukaryotes. We performed an assay using two complementary methods, pyrosequencing and clone library screening. In doing this, specificities for the group (ciliates) targeted in this study in bulked environmental samples were 94.6% for the clone library and 99.2% for pyrosequencing, respectively. In particular, our novel technique showed high selectivity for rare species, a feature that may be more important than the ability to identify quantitatively predominant species in community structure analyses. Additionally, our data revealed that a target-specific library (or ciliate-specific one for the present study) can better explain the ecological features of a sampling locality than a universal library.

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Phylum- and Class-Specific PCR Primers for General Microbial Community Analysis

Cited by 99 publications

References 41 publications

Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities

Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities

Assessment of vaginal microbiota in Brazilian women with and without bacterial vaginosis and comparison with Nugent score

Development of Single-Nucleotide Polymorphism-Based Phylum-Specific PCR Amplification Technique: Application to the Community Analysis Using Ciliates as a Reference Organism

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