P1411 PCR with universal primers targeting the small ribosomal RNA (16S rRNA) gene of bacteria as a diagnostic tool in 15 hospitalised patients with infectious diseases

Strains of Desulfitobacterium hafniense, such as strains PCP-1, DP7, TCE1, and TCP-A, have unusual long 16S ribosomal RNA (rRNA) genes due to an insertion of approximately 100 bp in the 5' region. In this report, we analyzed the 16S rRNA genes of different Desulfitobacterium strains to determine if such an insertion is a common feature of desulfitobacteria. We amplified this region by polymerase chain reaction (PCR) from eight Desulfitobacterium strains (D. hafniense strains PCP-1, DP7, TCP-A, TCE1, and DCB-2; D. dehalogenans; D. chlororespirans; and Desulfitobacterium sp. PCE1) and resolved each PCR product by denaturing gradient gel electrophoresis (DGGE). All strains had from two to seven DGGE- migrating bands, suggesting heterogeneity in their 16S rRNA gene copies. For each strain, the 5' region of the 16S rRNA genes was amplified and a clone library was derived. Clones corresponding to most PCR–DGGE migration bands were isolated. Sequencing of representative clones revealed that the heterogeneity was generated by insertions of 100–200 bp. An insertion was found in at least one copy of the 16S rRNA gene in all examined strains. In total, we found eight different types of insertions (INS1–INS8) that varied from 123 to 193 nt in length. Two-dimensional structural analyses of transcribed sequences predicted that all insertions would form an energetically stable loop. Reverse transcriptase – PCR experiments revealed that most of the observed insertions in the Desulfitobacterium strains were excised from the mature 16S rRNA transcripts. Insertions were not commonly found in bacterial 16S rRNA genes, and having a different insertion in several 16S rRNA gene copies borne by a single bacterial species was rarely observed. The function of these insertions is not known, but their occurrence can have an important impact in deriving 16S rRNA oligonucleotidic fluorescence in situ hybridization probes, as these insertions can be excised from 16S rRNA transcripts.Key words: Desulfitobacterium, 16S ribosomal RNA genes, heterogeneity, gene insertions, fluorescence in situ hybridization.

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

Scientific Reports ◽

10.1038/srep16498 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 69

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.

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KAJIAN MOLEKULER PADA PROBIOTIK ASAL AIR SUSU IBU DALAM SINTESIS EKSOPOLISAKARIDA (EPS)

Jurnal Bioteknologi & Biosains Indonesia (JBBI) ◽

10.29122/jbbi.v8i1.4554 ◽

2021 ◽

Vol 8 (1) ◽

pp. 114-123

Author(s):

Hamiyawati Qoimatu Dini Alfaruqi ◽

Nosa Septiana Anindita ◽

Arif Bimantara

Keyword(s):

Breast Milk ◽

16S Rrna ◽

16S Rrna Gene ◽

Universal Primers ◽

Rrna Gene ◽

Human Breast Milk ◽

Human Breast ◽

Adhesion Ability ◽

The 16S Rrna Gene ◽

Selection Of

Molecular Studies on Probiotic of Human Breast Milk in the Synthesis of Exopolysaccharide (EPS) The glucosyltransferase (gtf) gene has an important role in exopolysaccharide (EPS) synthesis in probiotic bacteria. The EPS produced is associated with the adhesion ability of bacteria to the intestinal mucosa. Therefore, the gtf gene can be used as a parameter in the selection of potential probiotic through a molecular approach. This study was conducted to determine the presence of the gtf gene in probiotic from human breast milk using PCR technique. The methods in this study include the following: reculture of probiotic isolates, DNA isolation, amplification of the 16S rRNA gene using universal primers (pA and pB), amplification of specific LAB primers (LABfw and LABrv), specific primary design for the gtf gene, and the amplification of the gtf gene. The results of 16S rRNA gene amplification using universal primers obtained the amplicons of 500-1,000 bp in size. The results of amplification using specific LAB primers obtained an amplicon of about 700 bp in all isolates. The results of amplification of the gtf gene using a specific primer produced an amplicon of 325 bp in all isolates. Based on this study, it was concluded that 16 probiotic isolates from human breast milk were proven to have the gtf gene. Gen glukosiltransferase (gtf) memiliki peran penting dalam sintesis eksopolisakarida (EPS) pada bakteri probiotik. EPS yang diproduksi berhubungan dengan kemampuan adhesi bakteri pada mukosa usus. Oleh karena itu, gen gtf dapat dijadikan sebagai salah satu parameter dalam seleksi probiotik potensial melalui pendekatan molekuler. Penelitian ini dilakukan untuk mengetahui adanya gen gtf pada probiotik asal air susu ibu (ASI) menggunakan teknik PCR. Metode pada penelitian ini meliputi: reculture isolat probiotik, isolasi DNA, amplifikasi gen 16S rRNA menggunakan primer universal (pA dan pB), amplifikasi primer spesifik BAL (LABfw dan LABrv), desain primer spesifik untuk gen gtf dan amplifikasi gen gtf. Hasil amplifikasi gen 16S rRNA menggunakan primer universal diperoleh amplikon berukuran antara 500-1.000 bp. Adapun hasil amplifikasi menggunakan primer spesifik BAL diperoleh amplikon berukuran sekitar 700 bp pada seluruh isolat. Hasil amplifikasi gen gtf menggunakan primer spesifik menghasilkan amplikon berukuran sekitar 325 bp pada seluruh isolat. Berdasarkan penelitian ini dapat disimpulkan bahwa 16 isolat probiotik asal ASI terbukti memiliki gen gtf.

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The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR

Brazilian Journal of Biology ◽

10.1590/1519-6984.247529 ◽

2023 ◽

Vol 83 ◽

Author(s):

A. Belmok ◽

T. Rodrigues-Oliveira ◽

F.A.C. Lopes ◽

R.H. Krüger ◽

C.M. Kyaw

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Sequences ◽

16S Rrna Genes ◽

Rrna Genes ◽

Universal Primers ◽

Rrna Gene ◽

Natural Habitats ◽

16S Rdna Pcr ◽

Archaeal Communities

Abstract Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

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Advantage of Using Inosine at the 3′ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity

Applied and Environmental Microbiology ◽

10.1128/aem.00849-06 ◽

2006 ◽

Vol 72 (11) ◽

pp. 6902-6906 ◽

Cited By ~ 61

Author(s):

Eitan Ben-Dov ◽

Orr H. Shapiro ◽

Nachshon Siboni ◽

Ariel Kushmaro

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Diversity ◽

Microbial Communities ◽

Environmental Samples ◽

Annealing Temperature ◽

Universal Primers ◽

Rrna Gene ◽

Bacterial Phyla ◽

Primer Sets

ABSTRACT To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3′ termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54°C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50°C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3′ termini in studying the microbial diversity of environmental samples.

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Rapid, one-step DNA extraction for the identification of German cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae) using DNA sequence of mitochondrial ribosomal RNA gene (16S rRNA gene)

Journal of Entomological Research ◽

10.5958/0974-4576.2020.00035.3 ◽

2020 ◽

Vol 44 (2) ◽

pp. 189

Author(s):

Ahmed Mahmood Rajab ◽

Gholamhossein Moravvej ◽

Ahmad Asoodeh

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Dna Extraction ◽

Dna Sequence ◽

Ribosomal Rna ◽

German Cockroach ◽

Rrna Gene ◽

Gene 16S Rrna ◽

One Step ◽

Cockroach Blattella Germanica

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Species-level resolution of 16S rRNA gene amplicons sequenced through the MinIONTM portable nanopore sequencer

10.1101/021758 ◽

2015 ◽

Cited By ~ 1

Author(s):

Alfonso Benítez-Páez ◽

Kevin J. Portune ◽

Yolanda Sanz

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Diversity ◽

Amplicon Sequencing ◽

Species Level ◽

Taxonomic Resolution ◽

Universal Primers ◽

Rrna Gene ◽

Rdna Sequences ◽

Dna Sequencer

AbstractBackgroundThe miniaturised and portable DNA sequencer MinIONTM has been released to the scientific community within the framework of an early access programme to evaluate its application for a wide variety of genetic approaches. This technology has demonstrated great potential, especially in genome-wide analyses. In this study, we tested the ability of the MinIONTM system to perform amplicon sequencing in order to design new approaches to study microbial diversity using nearly full-length 16S rDNA sequences.ResultsUsing R7.3 chemistry, we generated more than 3.8 million events (nt) during a single sequencing run. These data were sufficient to reconstruct more than 90% of the 16S rRNA gene sequences for 20 different species present in a mock reference community. After read mapping and 16S rRNA gene assembly, consensus sequences and 2d reads were recovered to assign taxonomic classification down to the species level. Additionally, we were able to measure the relative abundance of all the species present in a mock community and detected a biased species distribution originating from the PCR reaction using ‘universal’ primers.ConclusionsAlthough nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, the MinIONTM DNA sequencer is valuable for both high taxonomic resolution and microbial diversity analysis. Improvements in nanopore chemistry, such as minimising base-calling errors and the nucleotide bias reported here for 16S amplicon sequencing, will further deliver more reliable information that is useful for the specific detection of microbial species and strains in complex ecosystems.

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