scholarly journals Novel Approach to Quantitative Detection of Specific rRNA in a Microbial Community, Using Catalytic DNA

2005 ◽  
Vol 71 (8) ◽  
pp. 4879-4884 ◽  
Author(s):  
Hikaru Suenaga ◽  
Rui Liu ◽  
Yuko Shiramasa ◽  
Takahiro Kanagawa
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Bacterial Species ◽  
Quantitative Detection ◽  
Catalytic Function ◽  
Novel Approach ◽  
Specific Manner ◽  
Novel Method ◽  
Species Specific ◽  
Wastewater Treatment Systems

ABSTRACT We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.

scholarly journals A high-resolution genomic composition-based method with the ability to distinguish similar bacterial organisms

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Yizhuang Zhou ◽  
Wenting Zhang ◽  
Huixian Wu ◽  
Kai Huang ◽  
Junfei Jin
Keyword(s):  
High Resolution ◽  
Pearson Correlation ◽  
Bacterial Species ◽  
Real Data ◽  
Species Differentiation ◽  
Similar Species ◽  
Novel Approach ◽  
Identical Composition ◽  
Novel Method ◽  
Species Specific

Abstract Background Genomic composition has been found to be species specific and is used to differentiate bacterial species. To date, almost no published composition-based approaches are able to distinguish between most closely related organisms, including intra-genus species and intra-species strains. Thus, it is necessary to develop a novel approach to address this problem. Results Here, we initially determine that the “tetranucleotide-derived z-value Pearson correlation coefficient” (TETRA) approach is representative of other published statistical methods. Then, we devise a novel method called “Tetranucleotide-derived Z-value Manhattan Distance” (TZMD) and compare it with the TETRA approach. Our results show that TZMD reflects the maximal genome difference, while TETRA does not in most conditions, demonstrating in theory that TZMD provides improved resolution. Additionally, our analysis of real data shows that TZMD improves species differentiation and clearly differentiates similar organisms, including similar species belonging to the same genospecies, subspecies and intraspecific strains, most of which cannot be distinguished by TETRA. Furthermore, TZMD is able to determine clonal strains with the TZMD = 0 criterion, which intrinsically encompasses identical composition, high average nucleotide identity and high percentage of shared genomes. Conclusions Our extensive assessment demonstrates that TZMD has high resolution. This study is the first to propose a composition-based method for differentiating bacteria at the strain level and to demonstrate that composition is also strain specific. TZMD is a powerful tool and the first easy-to-use approach for differentiating clonal and non-clonal strains. Therefore, as the first composition-based algorithm for strain typing, TZMD will facilitate bacterial studies in the future.

scholarly journals Phylogenetic Diversity and Molecular Detection of Bacteria in Gull Feces

2008 ◽  
Vol 74 (13) ◽  
pp. 3969-3976 ◽  
Author(s):  
Jingrang Lu ◽  
Jorge W. Santo Domingo ◽  
Regina Lamendella ◽  
Thomas Edge ◽  
Stephen Hill
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Diversity ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Fecal Dna ◽  
Environmental Waters ◽  
Potential Risks ◽  
Species Specific ◽  
Gut Microbial Community

ABSTRACT In spite of increasing public health concerns about the potential risks associated with swimming in waters contaminated with waterfowl feces, little is known about the composition of the gut microbial community of aquatic birds. To address this, a gull 16S rRNA gene clone library was developed and analyzed to determine the identities of fecal bacteria. Analysis of 282 16S rRNA gene clones demonstrated that the gull gut bacterial community is mostly composed of populations closely related to Bacilli (37%), Clostridia (17%), Gammaproteobacteria (11%), and Bacteriodetes (1%). Interestingly, a considerable number of sequences (i.e., 26%) were closely related to Catellicoccus marimammalium, a gram-positive, catalase-negative bacterium. To determine the occurrence of C. marimammalium in waterfowl, species-specific 16S rRNA gene PCR and real-time assays were developed and used to test fecal DNA extracts from different bird (n = 13) and mammal (n = 26) species. The results showed that both assays were specific to gull fecal DNA and that C. marimammalium was present in gull fecal samples collected from the five locations in North America (California, Georgia, Ohio, Wisconsin, and Toronto, Canada) tested. Additionally, 48 DNA extracts from waters collected from six sites in southern California, Great Lakes in Michigan, Lake Erie in Ohio, and Lake Ontario in Canada presumed to be impacted with gull feces were positive by the C. marimammalium assay. Due to the widespread presence of this species in gulls and environmental waters contaminated with gull feces, targeting this bacterial species might be useful for detecting gull fecal contamination in waterfowl-impacted waters.

scholarly journals A new method for quantitative detection of Lactobacillus casei based on casx gene and its application

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xiaoyang Pang ◽  
Ziyang Jia ◽  
Jing Lu ◽  
Shuwen Zhang ◽  
Cai Zhang ◽  
...  
Keyword(s):  
16S Rrna ◽  
Detection Method ◽  
High Sensitivity ◽  
Accurate Method ◽  
Quantitative Detection ◽  
Bacterial Identification ◽  
Associated Proteins ◽  
16S Sequence ◽  
Species Specific ◽  
16 S Rrna

Abstract Background The traditional method of bacterial identification based on 16S rRNA is a widely used and very effective detection method, but this method still has some deficiencies, especially in the identification of closely related strains. A high homology with little differences is mostly observed in the 16S sequence of closely related bacteria, which results in difficulty to distinguish them by 16S rRNA-based detection method. In order to develop a rapid and accurate method of bacterial identification, we studied the possibility of identifying bacteria with other characteristic fragments without the use of 16S rRNA as detection targets. Results We analyzed the potential of using cas (CRISPR-associated proteins) gene as a target for bacteria detection. We found that certain fragment located in the casx gene was species-specific and could be used as a specific target gene. Based on these fragments, we established a TaqMan MGB Real-time PCR method for detecting bacteria. We found that the method used in this study had the advantages of high sensitivity and good specificity. Conclusions The casx gene-based method of bacterial identification could be used as a supplement to the conventional 16 s rRNA-based detection method. This method has an advantage over the 16 s rRNA-based detection method in distinguishing the genetic relationship between closely-related bacteria, such as subgroup bacteria, and can be used as a supplement to the 16 s rRNA-based detection method.

scholarly journals Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2565-2573 ◽  
Author(s):  
Xia Zhou ◽  
Stephen J. Bent ◽  
Maria G. Schneider ◽  
Catherine C. Davis ◽  
Mohammed R. Islam ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Dna Sequences ◽  
Phylogenetic Analyses ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Single Species ◽  
Rrna Gene ◽  
Caucasian Women ◽  
Tract Infections

The normal microbial flora of the vagina plays an important role in preventing genital and urinary tract infections in women. Thus an accurate understanding of the composition and ecology of the ecosystem is important to understanding the aetiology of these diseases. Common wisdom is that lactobacilli dominate the normal vaginal microflora of post-pubertal women. However, this conclusion is based on methods that require cultivation of microbial populations; an approach that is known to yield a biased and incomplete assessment of microbial community structure. In this study cultivation-independent methods were used to analyse samples collected from the mid-vagina of five normal healthy Caucasian women between the ages of 28 and 44. Total microbial community DNA was isolated following resuspension of microbial cells from vaginal swabs. To identify the constituent numerically dominant populations in each community 16S rRNA gene libraries were prepared following PCR amplification using the 8f and 926r primers. From each library, the DNA sequences of approximately 200 16S rRNA clones were determined and subjected to phylogenetic analyses. The diversity and kinds of organisms that comprise the vaginal microbial community varied among women. Species of Lactobacillus appeared to dominate the communities in four of the five women. However, the community of one woman was dominated by Atopobium sp., whereas a second woman had appreciable numbers of Megasphaera sp., Atopobium sp. and Leptotrichia sp., none of which have previously been shown to be common members of the vaginal ecosystem. Of the women whose communities were dominated by lactobacilli, there were two distinct clusters, each of which consisted of a single species. One class consisted of two women with genetically divergent clones that were related to Lactobacillus crispatus, whereas the second group of two women had clones of Lactobacillus iners that were highly related to a single phylotype. These surprising results suggest that culture-independent methods can provide new insights into the diversity of bacterial species found in the human vagina, and this information could prove to be pivotal in understanding risk factors for various infectious diseases.

scholarly journals Species-Specific PCR for Identification of Common Contaminant Mollicutes in Cell Culture

2001 ◽  
Vol 67 (7) ◽  
pp. 3195-3200 ◽  
Author(s):  
Fanrong Kong ◽  
Gregory James ◽  
Susanna Gordon ◽  
Anna Zelynski ◽  
Gwendolyn L. Gilbert
Keyword(s):  
16S Rrna ◽  
Cell Cultures ◽  
Bacterial Species ◽  
Intergenic Spacer ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Species Specific

ABSTRACT Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5′ end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5′ end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.

scholarly journals PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor

Biosensors ◽  
2016 ◽  
Vol 6 (3) ◽  
pp. 37 ◽  
Author(s):  
Leyla Esfandiari ◽  
Siqing Wang ◽  
Siqi Wang ◽  
Anisha Banda ◽  
Michael Lorenzini ◽  
...  
Keyword(s):  
16S Rrna ◽  
Bacterial Species ◽  
Pore Blockage ◽  
Species Specific

scholarly journals Polymerase chain reaction in the identification of periodontopathogens: A reliable and satisfactory method?

2012 ◽  
Vol 64 (4) ◽  
pp. 1413-1423
Author(s):  
Natasa Nikolic-Jakoba ◽  
Sandra Vojnovic ◽  
A. Pavic ◽  
S. Jankovic ◽  
V. Lekovic ◽  
...  
Keyword(s):  
16S Rrna ◽  
Bacterial Species ◽  
In Silico Analysis ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Conventional Pcr ◽  
Universal Primer ◽  
Health And Disease ◽  
Rrna Gene Sequencing ◽  
Species Specific

Aggregatibacter actinomycetemcomitans is considered one of the bacterial species of etiological importance in periodontitis. The aim of this study was to evaluate the serotype of A. actinomycetemcomitans in the subgingival biofilm in subjects with periodontal health and disease. Pooled samples of subgingival plaque were taken for culture-based identification of microorganisms. Colonies suspected to be A. actinomycetemcomitans were selected for molecular identification using either multiplex or conventional PCR in serotype-specific genotyping and 16S rRNA gene sequencing. In silico analysis showed that most selected colonies belong to the genus Campylobacter, although positive signals for serotypes of A. actinomycetemcomitans were obtained with these samples. Identification of A. actinomycetemcomitans by conventional PCR for 16S rRNA with one species-specific and one universal primer was inconclusive because an almost identical signal with Campylobacter gracilis was obtained. Although PCR-based methods for the identification of A. actinomycetemcomitans are more rapid, sequencing should not be omitted.

scholarly journals The structured diversity of specialized gut symbionts of the New World army ants

2016 ◽  
Author(s):  
Piotr Łukasik ◽  
Justin A. Newton ◽  
Jon G. Sanders ◽  
Yi Hu ◽  
Corrie S. Moreau ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
New World ◽  
Bacterial Species ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Army Ants ◽  
Army Ant ◽  
Species Specific

Symbiotic bacteria play important roles in the biology of their arthropod hosts. Yet the microbiota of many diverse and influential groups remain understudied, resulting in a paucity of information on the fidelities and histories of these associations. Motivated by prior findings from a smaller scale, 16S rRNA-based study, we conducted a broad phylogenetic and geographical survey of microbial communities in the ecologically dominant New World army ants (Formicidae: Dorylinae). Amplicon sequencing of the 16S rRNA gene across 28 species spanning the five New World genera showed that the microbial communities of army ants consist of very few common and abundant bacterial species. The two most abundant microbes, referred to as Unclassified Firmicutes and Unclassified Entomoplasmatales, appear to be specialized army ant associates that dominate microbial communities in the gut lumen of three host genera, Eciton, Labidus and Nomamyrmex. Both are present in other army ant genera, including those from the Old World, suggesting that army ant symbioses date back to the Cretaceous. Extensive sequencing of bacterial protein-coding genes revealed multiple strains of these symbionts co-existing within colonies, but seldom within the same individual ant. Bacterial strains formed multiple host species-specific lineages on phylogenies, which often grouped strains from distant geographic locations. These patterns deviate from those seen in other social insects, and raise intriguing questions about the influence of army ant colony swarm-founding and within-colony genetic diversity on strain co-existence, and the effects of hosting a diverse suite of symbiont strains on colony ecology.

scholarly journals PaSiT: a novel approach based on short-oligonucleotide frequencies for efficient bacterial identification and typing

2020 ◽  
Vol 36 (8) ◽  
pp. 2337-2344 ◽  
Author(s):  
Gleb Goussarov ◽  
Ilse Cleenwerck ◽  
Mohamed Mysara ◽  
Natalie Leys ◽  
Pieter Monsieurs ◽  
...  
Keyword(s):  
Large Scale ◽  
Bacterial Species ◽  
Supplementary Information ◽  
Nucleotide Identity ◽  
Average Nucleotide Identity ◽  
Bacterial Genomes ◽  
Short Oligonucleotide ◽  
Novel Approach ◽  
Novel Method ◽  
Alignment Step

Abstract Motivation One of the most widespread methods used in taxonomy studies to distinguish between strains or taxa is the calculation of average nucleotide identity. It requires a computationally expensive alignment step and is therefore not suitable for large-scale comparisons. Short oligonucleotide-based methods do offer a faster alternative but at the expense of accuracy. Here, we aim to address this shortcoming by providing a software that implements a novel method based on short-oligonucleotide frequencies to compute inter-genomic distances. Results Our tetranucleotide and hexanucleotide implementations, which were optimized based on a taxonomically well-defined set of over 200 newly sequenced bacterial genomes, are as accurate as the short oligonucleotide-based method TETRA and average nucleotide identity, for identifying bacterial species and strains, respectively. Moreover, the lightweight nature of this method makes it applicable for large-scale analyses. Availability and implementation The method introduced here was implemented, together with other existing methods, in a dependency-free software written in C, GenDisCal, available as source code from https://github.com/LM-UGent/GenDisCal. The software supports multithreading and has been tested on Windows and Linux (CentOS). In addition, a Java-based graphical user interface that acts as a wrapper for the software is also available. Supplementary information Supplementary data are available at Bioinformatics online.

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