scholarly journals Development of a simple, rapid multiplex PCR tool kit by using 16S rRNA gene for the identification of faecal and non-faecal coli forms in the drinking waters

2021 ◽  
Author(s):  
A. Shiva Shanker ◽  
N. Rajesh ◽  
Pavan Kumar Pindi
Keyword(s):  
Drinking Water ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Multiplex Pcr ◽  
Faecal Coliform ◽  
Rrna Gene ◽  
Faecal Coliforms ◽  
Variable Regions ◽  
Total Coliforms ◽  
Multiplex Pcr Assay

Abstract A multiplex method for the detection of faecal and non-faecal coliforms in drinking water was developed using three primers from the V2, V3 and V9 variable regions of 16S rRNA gene. 194F, 474F and 1436R are the three primers designed for specific amplification of V2, V3, V9 hyper variable regions of 16S rRNA gene. Multiplex PCR allowed for differentiation of the total coliform from faecal coliform by specific amplicons: 1,285 bp of amplicon is specific for 6 non-faecal coliform genera and 1,009 bp of amplicon is specific for faecal coliform ie. E. coli. If the drinking water was contaminated with both faecal and non-faecal coliforms then two amplicons of 1,285 bp and 1,009 bp by combination of three primers are observed. The multiplex PCR assay based on 16S rRNA gene should be a beneficial tool kit for the rapid identification of the total coliforms in the large number of water samples compared with traditional methods. Results can be acquired within 3 hrs of time as compared with classic method of MPN (3–4 days). This assay will be useful in diversification and detection of seven genera of total coliforms by using variable regions of 16S rRNA.

scholarly journals Nocardioides hungaricus sp. nov., isolated from a drinking water supply system

2011 ◽  
Vol 61 (3) ◽  
pp. 549-553 ◽  
Author(s):  
Erika M. Tóth ◽  
Zsuzsa Kéki ◽  
Judit Makk ◽  
Zalán G. Homonnay ◽  
Károly Márialigeti ◽  
...  
Keyword(s):  
Drinking Water ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Supply ◽  
Gene Sequence ◽  
Supply System ◽  
Drinking Water Supply ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Drinking Water Supply System

Three Gram-positive, rod-shaped bacterial strains were isolated from the drinking water supply system of the Hungarian capital, Budapest. Phylogenetic analysis on the basis of 16S rRNA gene sequence comparison revealed that the isolates represented a distinct cluster within the clade of the genus Nocardioides and were most closely related to Nocardioides pyridinolyticus OS4T, Nocardioides aquiterrae GW-9T, Nocardioides sediminis MSL-01T and N. hankookensis DS-30T. The peptidoglycan based on ll-2,6-diaminopimelic acid, the major menaquinone MK-8(H4), the cellular fatty acid profile with iso-C16 : 0 and anteiso-C17 : 0 as predominating components and the DNA G+C content of 71.4 mol% (strain 1RaM5-12T) were consistent with the affiliation of the isolates to the genus Nocardioides. Because of differences in physiological characteristics, matrix-assisted laser-desorption/ionization time-of-flight mass spectra of protein extracts, PvuII RiboPrinter patterns and 96.1 % 16S rRNA gene sequence similarity between strain 1RaM5-12T and its closest phylogenetic neighbour, N. pyridinolyticus OS4T, a novel species, Nocardioides hungaricus sp. nov., is proposed. The type strain is 1RaM5-12T (=DSM 21673T =NCAIM 02330T).

scholarly journals Bacillus purgationiresistans sp. nov., isolated from a drinking-water treatment plant

2012 ◽  
Vol 62 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Ivone Vaz-Moreira ◽  
Vânia Figueira ◽  
Ana R. Lopes ◽  
Alexandre Lobo-da-Cunha ◽  
Cathrin Spröer ◽  
...  
Keyword(s):  
Drinking Water ◽  
Water Treatment ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Treatment Plant ◽  
Drinking Water Treatment ◽  
Water Treatment Plant ◽  
Rrna Gene ◽  
Rrna Gene Sequence

A Gram-positive, aerobic, non-motile, endospore-forming rod, designated DS22T, was isolated from a drinking-water treatment plant. Cells were catalase- and oxidase-positive. Growth occurred at 15–37 °C, at pH 7–10 and with <8 % (w/v) NaCl (optimum growth: 30 °C, pH 7–8 and 1–3 % NaCl). The major respiratory quinone was menaquinone 7, the G+C content of the genomic DNA was 36.5 mol% and the cell wall contained meso-diaminopimelic acid. On the basis of 16S rRNA gene sequence analysis, strain DS22T was a member of the genus Bacillus. Its closest phylogenetic neighbours were Bacillus horneckiae NRRL B-59162T (98.5 % 16S rRNA gene sequence similarity), Bacillus oceanisediminis H2T (97.9 %), Bacillus infantis SMC 4352-1T (97.4 %), Bacillus firmus IAM 12464T (96.8 %) and Bacillus muralis LMG 20238T (96.8 %). DNA–DNA hybridization, and biochemical and physiological characterization allowed the differentiation of strain DS22T from its closest phylogenetic neighbours. The data supports the proposal of a novel species, Bacillus purgationiresistans sp. nov.; the type strain is DS22T ( = DSM 23494T = NRRL B-59432T = LMG 25783T).

scholarly journals Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Kirsten A. Ziesemer ◽  
Allison E. Mann ◽  
Krithivasan Sankaranarayanan ◽  
Hannes Schroeder ◽  
Andrew T. Ozga ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Amplification ◽  
Amplicon Sequencing ◽  
Universal Primers ◽  
Rrna Gene ◽  
Dental Calculus ◽  
Shotgun Metagenomics ◽  
Variable Regions ◽  
V3 Region

Abstract To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

Genetic profiling of noncultivated bacteria from the rhizospheres of sugar beet (Beta vulgaris) reveal field and annual variability but no effect of a transgenic herbicide resistance

2003 ◽  
Vol 49 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Achim Schmalenberger ◽  
Christoph C Tebbe
Keyword(s):  
16S Rrna ◽  
Sugar Beet ◽  
16S Rrna Gene ◽  
Beta Vulgaris ◽  
Herbicide Resistance ◽  
Microbial Community Analysis ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Variable Regions ◽  
Genetic Profiling

In this field study, we compared the bacterial communities inhabiting the rhizosphere of a transgenic, herbicide-resistant sugar beet (Beta vulgaris) cultivar with those of its nonengineered counterpart, using a genetic profiling technique based on PCR amplifications of partial 16S rRNA gene sequences and single-strand conformation polymorphism (SSCP). As a control for the plasticity of the bacterial community, we also analyzed the influence of herbicides, the field heterogeneity, and the annual variation. DNA was isolated from bacterial cell consortia that were directly collected from root material. PCR was carried out with primers that hybridized to evolutionarily conserved regions flanking variable regions 4 and 5 of the 16S rRNA gene. SSCP patterns of these PCR products were composed of approximately 50 distinguishable bands, as detected by silver staining of the gels after electrophoresis. Patterns of the replicates and the different treatments were highly similar, but digital image and similarity analyses revealed differences that corresponded to the positions of the replicates in the field. In addition, communities collected from sugar beet in two successive growing seasons could be distinguished. In contrast, no effect of the transgenic herbicide resistance was detectable. Sequencing of 24 dominant products of the SSCP profiles indicated the presence of bacteria from different phylogenetic groups, with Proteobacteria and members of the Cytophaga–Flavobacterium–Bacteroides group being most abundant.Key words: genetic profiles, rRNA genes, transgenic sugar beet, risk assessment, rhizosphere, PCR–SSCP, microbial community analysis, glufosinate, phosphinothricin.

scholarly journals Combining 16S rRNA gene variable regions enables high-resolution microbial community profiling

2017 ◽  
Author(s):  
Garold Fuks ◽  
Michael Elgart ◽  
Amnon Amir ◽  
Amit Zeisel ◽  
Peter J. Turnbaugh ◽  
...  
Keyword(s):  
High Resolution ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Read Length ◽  
Rrna Gene ◽  
Sample Number ◽  
Variable Regions ◽  
Total Length ◽  
Fragmented Dna ◽  
The 16S Rrna Gene

AbstractBackgroundMost of our knowledge about the remarkable microbial diversity on Earth comes from sequencing the 16S rRNA gene. The use of next-generation sequencing methods has increased sample number and sequencing depth, but the read length of the most widely used sequencing platforms today is quite short, requiring the researcher to choose a subset of the gene to sequence (typically 16-33% of the total length). Thus, many bacteria may share the same amplified region and the resolution of profiling is inherently limited. Platforms that offer ultra long read lengths, whole genome shotgun sequencing approaches, and computational frameworks formerly suggested by us and by others, all allow different ways to circumvent this problem yet suffer various shortcomings. There is need for a simple and low cost 16S rRNA gene based profiling approach that harnesses the short read length to provide a much larger coverage of the gene to allow for high resolution, even in harsh conditions of low bacterial biomass and fragmented DNA.ResultsThis manuscript suggests Short MUltiple Regions Framework (SMURF), a method to combine sequencing results from different PCR-amplified regions to provide one coherent profiling. The de facto amplicon length is the total length of all amplified regions, thus providing much higher resolution compared to current techniques. Computationally, the method solves a convex optimization problem that allows extremely fast reconstruction and requires only moderate memory. We demonstrate the increase in resolution by in silico simulations and by profiling two mock mixtures and real-world biological samples. Reanalyzing a mock mixture from the Human Microbiome Project achieved about two-fold improvement in resolution when combing two independent regions. Using a custom set of six primer pairs spanning about 1200bp (80%) of the 16S rRNA gene we were able to achieve ~100 fold improvement in resolution compared to a single region, over a mock mixture of common human gut bacterial isolates. Finally, profiling of a Drosophila melanogaster microbiome using the set of six primer pairs provided a ~100 fold increase in resolution, and thus enabling efficient downstream analysis.ConclusionsSMURF enables identification of near full-length 16S rRNA gene sequences in microbial communities, having resolution superior compared to current techniques. It may be applied to standard sample preparation protocols with very little modifications. SMURF also paves the way to high-resolution profiling of low-biomass and fragmented DNA, e.g., in the case of Formalin-fixed and Paraffin-embedded samples, fossil-derived DNA or DNA exposed to other degrading conditions. The approach is not restricted to combining amplicons of the 16S rRNA gene and may be applied to any set of amplicons, e.g., in Multilocus Sequence Typing (MLST).

scholarly journals Resolving microbial microdiversity with high accuracy full length 16S rRNA Illumina sequencing

2014 ◽  
Author(s):  
Catherine Burke ◽  
Aaron E Darling
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Single Molecule ◽  
Illumina Miseq ◽  
Full Length ◽  
Rrna Gene ◽  
Variable Regions ◽  
Phylogenetic Resolution ◽  
Miseq Platform ◽  
The 16S Rrna Gene

We describe a method for sequencing full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform. The resulting sequences have about 100-fold higher accuracy than standard Illumina reads and are chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection. We demonstrate that the data provides fine scale phylogenetic resolution not available from Illumina amplicon methods targeting smaller variable regions of the 16S rRNA gene.

scholarly journals Combining 16S rRNA gene variable regions enables high-resolution microbial community profiling

Microbiome ◽  
2018 ◽  
Vol 6 (1) ◽  
Author(s):  
Garold Fuks ◽  
Michael Elgart ◽  
Amnon Amir ◽  
Amit Zeisel ◽  
Peter J. Turnbaugh ◽  
...  
Keyword(s):  
Microbial Community ◽  
High Resolution ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Variable Regions ◽  
Microbial Community Profiling ◽  
Community Profiling
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