Universal Bacterial Identification by PCR and DNA Sequencing of 16S rRNA Gene

2010 ◽  
pp. 209-214 ◽  
Author(s):  
Greg James
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequencing ◽  
Rrna Gene ◽  
Bacterial Identification

scholarly journals Astacopsidrilus hibernicus sp. nov. (Phreodrilidae, Oligochaeta, Annelida) from Irish peatlands

Zoosymposia ◽  
2020 ◽  
Vol 17 (1) ◽  
pp. 34-44
Author(s):  
RÜDIGER M. SCHMELZ ◽  
MÅRTEN J. KLINTH ◽  
RACHEL WISDOM ◽  
THOMAS BOLGER
Keyword(s):  
United Kingdom ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequencing ◽  
Dorsal Side ◽  
Rrna Gene ◽  
The United Kingdom ◽  
The 16S Rrna Gene ◽  
Epidermal Gland ◽  
Whole Mounts

The discovery of a large and flourishing population of Phreodrilidae in terrestrial peatlands in northwest Ireland was surprising on two counts: these oligochaete worms are usually aquatic and most of the species occur in the Southern Hemisphere. The phreodrilids were discovered in a project that targeted Enchytraeidae, therefore methods adapted to the investigation of enchytraeids could be applied, including the study of living animals and properly fixed whole mounts. DNA sequencing was also performed. All worms identified here belong to one species, new to science, and placed in the genus Astacopsidrilus, because of the ventral position of the spermathecal pores and the opening of the female funnels inside the spermathecal vestibule. Astacopsidrilus hibernicus sp. nov. is mainly distinguished by thick segmental cushions of epidermal gland cells on the dorsal side of the posterior body half. Male sexual organs and spermathecae are comparatively small and without the often-observed bizarre modifications common in species of this family. DNA sequencing yielded a fragment of the 16S rRNA gene. This is the first description of a phreodrilid species from Europe; the few previous recordings of this family in Ireland and the United Kingdom had been left unidentified.

Molecular Identification of Commercial Fish Maws using mtDNA 16S rRNA Nucleotide Sequences

2020 ◽  
Author(s):  
Jinju Zhang ◽  
Qiuting Deng ◽  
Nanxin Li ◽  
Xindan Liu ◽  
Menghua Wu ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequencing ◽  
Fish Species ◽  
Molecular Characteristics ◽  
Rrna Gene ◽  
Commercial Fish ◽  
Interspecific Divergence ◽  
Model Sample ◽  
Original Species

Abstract Background: Fish maws (dried swim bladders of fish) have long been used as a medicinal material and valuable tonic food which are very popular in Southeast Asia. It is difficult to identify the original species of fish maws sold on the market due to a lack of taxonomic characteristics. The present study aims to investigate the origin of commercial fish maws using DNA sequencing based on a partial sequence of the mtDNA 16S rRNA gene.Methods: Mitochondrial genomic DNA was extracted from a total of 44 individual fish maw samples collected from different markets by a QIAamp® DNA Mini Kit. The fragments of the 16S rRNA gene were amplified by PCR and bidirectionally sequenced using an ABI 3730 genetic analyser. Sequence assembly was performed with DNASTAR SeqMan software, and the resulting sequence was compared with reference sequences in GenBank using BLAST. Interspecific divergence and intraspecific variation were calculated by the Kimura 2-parameter model. Sample sequences were clustered in MEGA 6 using a neighbour-joining tree.Results: Forty-two of 44 fish maw samples were sequenced successfully. Fourteen taxa matched known fish species in the DNA sequence database, and 10 of them were supported by high homogeneity (99-100%). According to the results, 88% of the total samples belonged to family Sciaenidae. The clustering and genetic distance results, including in-group divergence and out-group divergence, indicated confidence in species identifications. Moreover, there was no strict correspondence between the commercial names provided by traders and the origin of the fish maws. This study also found a probable correlation between the molecular characteristics and morphological features of fish maws from croakers and non-croakers.Conclusion: In this study, DNA sequencing based on mtDNA 16S rRNA was carried out successfully to identify the origin of ~95% of the samples. This result indicated that 16S rRNA is a suitable barcode for identifying fish maws. The identification results allowed us to learn more about the fish species available in the fish maw market in Guangdong-Hongkong-Macao Great Bay. In addition, the association between trade category and species found in this study provides both consumers and merchants with an important reference for identifying the origin of fish maws.

Intraspecific variation in 16S rRNA gene of Mycoplasma synoviae determined by DNA sequencing

2010 ◽  
Vol 33 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Marcos R. Buim ◽  
Melissa Buzinhani ◽  
Maurício Yamaguti ◽  
Rosângela C. Oliveira ◽  
Elena Mettifogo ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequencing ◽  
Intraspecific Variation ◽  
Rrna Gene ◽  
Mycoplasma Synoviae

scholarly journals Rapid bacterial identification by direct PCR amplification of 16S rRNA genes using the MinIONTMnanopore sequencer

2018 ◽  
Author(s):  
Shinichi Kai ◽  
Yoshiyuki Matsuo ◽  
So Nakagawa ◽  
Kirill Kryukov ◽  
Shino Matsukawa ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bacterial Identification ◽  
Direct Pcr ◽  
16S Rrna Gene Analysis ◽  
Bacterial Dna

AbstractRapid identification of bacterial pathogens is crucial for appropriate and adequate antibiotic treatment, which significantly improves patient outcomes. 16S ribosomal RNA (rRNA) gene amplicon sequencing has proven to be a powerful strategy for diagnosing bacterial infections. We have recently established a sequencing method and bioinformatics pipeline for 16S rRNA gene analysis utilizing the Oxford Nanopore Technologies MinION™ sequencer. In combination with our taxonomy annotation analysis pipeline, the system enabled the molecular detection of bacterial DNA in a reasonable timeframe for diagnostic purposes. However, purification of bacterial DNA from specimens remains a rate-limiting step in the workflow. To further accelerate the process of sample preparation, we adopted a direct PCR strategy that amplifies 16S rRNA genes from bacterial cell suspensions without DNA purification. Our results indicate that differences in cell wall morphology significantly affect direct PCR efficiency and sequencing data. Notably, mechanical cell disruption preceding direct PCR was indispensable for obtaining an accurate representation of the specimen bacterial composition. Furthermore, 16S rRNA gene analysis of mock polymicrobial samples indicated that primer sequence optimization is required to avoid preferential detection of particular taxa and to cover a broad range of bacterial species. This study establishes a relatively simple workflow for rapid bacterial identification via MinIONTMsequencing, which reduces the turnaround time from sample to result, and provides a reliable method that may be applicable to clinical settings.

scholarly journals Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

2016 ◽  
Author(s):  
Patrick D Schloss ◽  
Matthew L Jenior ◽  
Charles C. Koumpouras ◽  
Sarah L Westcott ◽  
Sarah K Highlander
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequencing ◽  
Error Rate ◽  
Full Length ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Mock Community ◽  
Sequencing Platforms ◽  
The 16S Rrna Gene

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality, but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

scholarly journals Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring

2015 ◽  
Vol 72 (11) ◽  
pp. 1962-1972 ◽  
Author(s):  
J. Vierheilig ◽  
D. Savio ◽  
R. E. Ley ◽  
R. L. Mach ◽  
A. H. Farnleitner ◽  
...  
Keyword(s):  
Water Quality ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequencing ◽  
Bacterial Communities ◽  
Bacterial Community Composition ◽  
Detection Methods ◽  
Rrna Gene ◽  
Next Generation ◽  
Next Generation Dna Sequencing

The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multi-compartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems.

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