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rRNA Primers (rrna + primer)
Selected AbstractsInfluence of long-term land application of Class B biosolids on soil bacterial diversityJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2010H. Zerzghi Abstract Aim:, To evaluate the effect of long-term annual land applications of Class B biosolids on soil bacterial diversity at University of Arizona Marana Agricultural Field Center, Tucson, Arizona. Methods and Results:, Following the final of 20 consecutive years of application of Class B biosolids in March 2005, followed by cotton growth from April to November 2005 surface soil samples (0,30 cm) were collected from control (unamended) and biosolid-amended plots. Total bacterial community DNA was extracted, amplified using 16S rRNA primers, cloned, and sequenced. All 16S rRNA sequences were identified by 16S rRNA sequence analysis and comparison to known sequences in GenBank (NCBI BlastN and Ribosomal Database Project II, RDP). Results showed that the number of known genera (identifiable > 96%) increased in the high rate biosolid plots compared to control plots. Biosolids-amended soils had a broad phylogenetic diversity comprising more than four major phyla: Proteobacteria (32%), Acidobacteria (21%), Actinobacteria (16%), Firmicutes (7%), and Bacteroidetes (6%) which were typical to bacterial diversity found in the unamended arid southwestern soils. Conclusion:, Bacterial diversity was either enhanced or was not negatively impacted following 20 years of land application of Class B biosolids. Significance and Impact of the Study:, This study illustrates that long-term land application of biosolids to arid southwestern desert soils has no deleterious effect on soil microbial diversity. [source] Diversity of aerosolized bacteria during land application of biosolidsJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2007J.P. Brooks Abstract Aims: The purpose of this study was to determine the diversity of bacterial communities associated with bioaerosols generated during land application of biosolids using 16S ribosomal RNA (16S rRNA) PCR. Methods and Results: Anaerobically digested Class B biosolids were land applied to an agricultural site located in South Central Arizona. Aerosol samples were collected downwind of the biosolids operations and were collected via the use of SKC Biosamplers and subsequently extracted for the presence of bacterial community DNA. All DNA was amplified using 16S rRNA primers, cloned and sequenced. All sequences were aligned and phylogenetic trees were developed to generate community profiles. The majority of aerosolized bacterial clone sequences belonged to the Actinobacteria and alpha - and beta - proteobacterial taxa. Aerosol samples collected downwind of soil aerosolization produced similar profiles. These profiles differed from upwind and background samples. Conclusions: No one clone sequence isolated from the aerosol samples could be solely attributed to biosolids; on the contrary, the majority appeared to have arisen from soil. Significance and Impact of the Study: This study demonstrates that in dry, arid climates the majority of aerosols associated with biosolids land application appear to be associated with the onsite soil. [source] Endpoint quantitative PCR assays for Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitansJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2003J. D. Rudney Background:, Conventional polymerase chain reaction (PCR) assays for periodontal pathogens are so sensitive that they detect infections of no clinical significance. Quantitative PCR (qPCR) may provide a solution to this problem. However, most qPCR systems require expensive real-time thermal cyclers. Objective:, Our goal was to develop qPCR assays which would allow endpoint quantification. Materials and methods:, 16S rRNA primers for Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans were adapted to the AmplifluorÔ qPCR system, which incorporates fluorescein into the PCR product so that endpoint fluorescence is proportional to the original amount of template. DNA dilutions representing known numbers of cells were used as standard curves. Pooled subgingival plaques from the four deepest pockets of 21 severe adult periodontitis patients were assayed. Buccal molar supragingival plaque from 35 dental students provided healthy controls. Endpoint fluorescence was measured with a microplate reader. Results:, Optimized standard curves were linear in log,log or semilog fits over a range of 100,106 cells. Countable B. forsythus was present in all patients, with counts (as logs) from 2.4 to 7.3 (mean = 5.0), and 11 controls with counts from 2.1 to 4.5 (mean = 3.0). P. gingivalis was present in 11 patients and no controls, with counts from 2.2 to 4.7 (mean = 3.2). A. actinomycetemcomitans was present in two patients, with counts of 1.5 and 3.5. Conclusions:, AmplifluorÔ qPCR assays discriminated between plaque samples differing by one log or more, allowing major infections to be distinguished from minor ones. This approach allows high-throughput qPCR of plaque samples, using equipment available to many laboratories. [source] Distribution of fimA genotypes of Porphyromonas gingivalis in subjects with various periodontal conditionsMOLECULAR ORAL MICROBIOLOGY, Issue 4 2004C. G. Missailidis Fimbria encoded by the gene fimA is considered one of the main factors in the colonization of the oral cavity by Porphyromonas gingivalis. Allelic variation in fimA led to the classification of strains of P. gingivalis into six genotypes. The occurrence of P. gingivalis was determined by polymerase chain reaction using 16S rRNA primers in 302 subgingival samples obtained from 102 Brazilian subjects exhibiting different periodontal conditions. Distribution of fimA genotypes was assessed in 146 P. gingivalis positive samples by polymerase chain reaction using primers pairs homologous to the different fimA genes. P. gingivalis was detected in 51 of 57 (89.4%) patients with periodontal attachment loss, in six of 20 gingivitis patients (30.0%) and in two of 25 (8.0%) subjects with a healthy periodontium. Variant type II was the only type detected in 53 sites (39.3%), distributed among 19 periodontitis patients (37.3%) and in one patient with no periodontal destruction. Type Ib was the second most prevalent genotype in periodontitis patients (19.6%). Genotype V was not detected in the studied population. Type IV was the most commonly type found among gingivitis patients, either alone or in combination with other genotypes. Multiple genotypes were detected in nine sites (6.1%). A fimA genotype was not identified in 26 sites (17.8%) of 146 sites positive for P. gingivalis, suggesting that other alleles of fimA not yet sequenced may be prevalent in this population. These data demonstrated that P. gingivalis type II strains followed by type Ib are more prevalent in periodontitis patients from a multiracial population in Brazil, suggesting an increased pathogenic potential of these types. [source] Procuration and identification of bacteria in paraffin-embedded liver tissues of hepatocellular carcinoma by laser-assisted microdissection technique,APMIS, Issue 1 2008XUE-FEI TIAN This study was aimed at procuring directly and identifying the bacteria which had been found in paraffin-embedded liver tissues of hepatocellular carcinoma (HCC) patients. In our previous studies, Helicobacter spp. had been detected by polymerase chain reaction (PCR) and observed by histology in the liver tissues of HCC patients but had never been cultured successfully. To obtain and identify the uncultured bacteria, laser microdissection and pressure catapulting (LMPC) techniques were applied. Following microdissection from the liver tissue sections, these bacteria were examined by PCR using Helicobacter genus-specific 16S rRNA primers and sequence analysis. Amplified products of 16S rRNA were positive in all six microdissected samples with bacteria, and showed 99%,100% similarity with Helicobacter pylori by sequence analysis. Another H. pylori -specific 26 kDa gene (encoding one 26 kDa protein as H. pylori- specific antigen) was also tested by PCR. Four of six samples were positive. Therefore, Helicobacter spp. detected by PCR in the liver tissues of HCC patients in our previous studies are actually the bacteria observed by histology and identified as H. pylori by further sequence analysis. The laser-assisted microdissection technique can be extensively applied for identification of bacteria in tissue samples in bacteriology research. [source] | ||||||||||