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Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays.

ABSTRACT: rRNA genes are attractive targets for developing PCR assays targeting human fungal pathogens. Most studies have focused on the 18S rRNA gene, internal transcribed spacers, and the 5' end of the 28S rRNA gene. An approximately 2,900-bp region of the 28S rRNA gene remains largely unexplored because sequences of many medically relevant fungi are either unavailable or undefined in genomic databases. The internal transcribed spacers and 28S rRNA gene of nine medically and phylogenetically important fungi were sequenced. In addition, 42 sequences from this region were acquired from public databases, resulting in an alignment of 51 fungal sequences spanning 30 fungal genera. For the nearly 3,950-bp region from the 3' end of 18S rRNA gene to the 3' end of the 28S rRNA gene, 27 broad-range PCR primers were designed such that their sequence homology with the human rRNA gene was minimal. All 62 possible amplicons in the size range from 75 to 400 bp from 27 primers were screened using fungal genomic DNA from 26 species spanning 14 genera. Eleven of the 62 amplicons did not cross-react with 1 microg/PCR human DNA but simultaneously amplified 10 fg of fungal DNA. Phylogenetic distance matrices were calculated for regions covered by these 11 amplicons based on 51 fungi. Two of these 11 amplicons successfully amplified 30 fg of fungal DNA from 25 of 26 fungi and provided the most phylogenetic information for species identification based on the distance matrices. These PCR assays hold promise for detection and identification of fungal pathogens in human tissues.

SUBMITTER: Khot PD

PROVIDER: S-EPMC2655463 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays.

Khot Prasanna D PD Ko Daisy L DL Fredricks David N DN

Applied and environmental microbiology 20090109 6

rRNA genes are attractive targets for developing PCR assays targeting human fungal pathogens. Most studies have focused on the 18S rRNA gene, internal transcribed spacers, and the 5' end of the 28S rRNA gene. An approximately 2,900-bp region of the 28S rRNA gene remains largely unexplored because sequences of many medically relevant fungi are either unavailable or undefined in genomic databases. The internal transcribed spacers and 28S rRNA gene of nine medically and phylogenetically important f ...[more]

PMID: 19139223

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Project description:INTRODUCTION: Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples. METHODS: We examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database. RESULTS: Bacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium genera incertae sedis OP10. CONCLUSIONS: Broad-range PCR followed by cloning and sequencing the entire 16S rDNA, allowed the identification of the bacterial DNA environment in the SF samples of arthritic patients. We found a wide spectrum of bacteria including those known to be involved in ReA and others not previously associated with arthritis.

Project description:INTRODUCTION: Bacteria and/or their antigens have been implicated in the pathogenesis of reactive arthritis (ReA). Several studies have reported the presence of bacterial antigens and nucleic acids of bacteria other than those specified by diagnostic criteria for ReA in joint specimens from patients with ReA and various arthritides. The present study was conducted to detect any bacterial DNA and identify bacterial species that are present in the synovial tissue of Tunisian patients with reactive arthritis and undifferentiated arthritis (UA) using PCR, cloning and sequencing. METHODS: We examined synovial tissue samples from 28 patients: six patients with ReA and nine with UA, and a control group consisting of seven patients with rheumatoid arthritis and six with osteoarthritis (OA). Using broad-range bacterial PCR producing a 1,400-base-pair fragment from the 16S rRNA gene, at least 24 clones were sequenced for each synovial tissue sample. To identify the corresponding bacteria, DNA sequences were compared with sequences from the EMBL (European Molecular Biology Laboratory) database. RESULTS: Bacterial DNA was detected in 75% of the 28 synovial tissue samples. DNA from 68 various bacterial species were found in ReA and UA samples, whereas DNA from 12 bacteria were detected in control group samples. Most of the bacterial DNAs detected were from skin or intestinal bacteria. DNA from bacteria known to trigger ReA, such as Shigella flexneri and Shigella sonnei, were detected in ReA and UA samples of synovial tissue and not in control samples. DNA from various bacterial species detected in this study have not previously been found in synovial samples. CONCLUSION: This study is the first to use broad-range PCR targeting the full 16S rRNA gene for detection of bacterial DNA in synovial tissue. We detected DNA from a wide spectrum of bacterial species, including those known to be involved in ReA and others not previously associated with ReA or related arthritis. The pathogenic significance of some of these intrasynovial bacterial DNAs remains unclear.

Project description:Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1. 4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.

Dataset Information

Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays.

Publications

Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays.

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