Overestimation of Streptococcus mutans prevalence by nested PCR detection of the 16S rRNA gene

2006 ◽  
Vol 55 (1) ◽  
pp. 109-113 ◽  
Author(s):  
Ali Al-Ahmad ◽  
Thorsten Mathias Auschill ◽  
Gabriele Braun ◽  
Elmar Hellwig ◽  
Nicole Birgit Arweiler
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Streptococcus Mutans ◽  
Nested Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Direct Pcr ◽  
Specific Primers ◽  
The 16S Rrna Gene

This study was carried out in order to compare two PCR-based methods in the detection of Streptococcus mutans. The first PCR method was based on primers for the 16S rRNA gene and the second method was based on specific primers that targeted the glucosyltransferase gene (gtfB). Each PCR was performed with eight different streptococci from the viridans group, five other streptococci and 17 different non-streptococcal bacterial strains. Direct use of the S. mutans 16S rRNA gene-specific primers revealed that Streptococcus gordonii and Streptococcus infantis were also detected. After amplifying the 16S rRNA gene with universal primers and subsequently performing nested PCR, the S. mutans-specific nested primers based on the 16S rRNA gene detected all tested streptococci. There was no cross-reaction of the gtfB primers after direct PCR. Our results indicate that direct PCR and nested PCR based on 16S rRNA genes can reveal false-positive results for oral streptococci and lead to an overestimation of the prevalence of S. mutans with regards to its role as the most prevalent causative agent of dental caries.

scholarly journals Comparison of P1 and 16S rRNA genes for detection of Mycoplasma pneumoniae by nested PCR in adults in Zhejiang, China

2015 ◽  
Vol 9 (03) ◽  
pp. 244-253 ◽  
Author(s):  
Zibo Zhou ◽  
Xiangzhi Li ◽  
Xiaojian Chen ◽  
Lili Yao ◽  
Changwang Pan ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Mycoplasma Pneumoniae ◽  
Nested Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Positive Rate ◽  
The 16S Rrna Gene

Introduction: Mycoplasma pneumoniae (M. pneumoniae) is the most common atypical pathogen that causes respiratory infections in humans. Laboratory tests are important in the diagnosis of M. pneumoniae because of the atypical features in clinical signs and symptoms. Nowadays, both the P1 adhesin gene and 16S ribosomal (r) RNA (rRNA) gene of M. pneumoniae have been widely detected by polymerase chain reaction (PCR). The purpose of the present study was to evaluate the most suitable target in the detection of M. pneumonia via nested PCR. Methodology: Both the P1 adhesin gene and 16S rRNA gene for nested PCR reaction conditions were optimized through an orthogonal test and single-factor experiment. Then, the sensitivity of the two sets of targets was evaluated. Finally, based on the optimal conditions, 55 clinical samples of throat swabs collected from adult patients in 2013 were examined by established nested PCR. Result: The results revealed that PCR detection of the 16S rRNA gene was more sensitive than the P1 adhesin gene because the detection limits for both the P1 gene and 16S rRNA gene were at least 100 fg and 10 fg of M. pneumoniae DNA, respectively. Furthermore, the positive rate for detection of the 16S rRNA gene (30/55; 54.5%) was higher than that of the P1 adhesin gene (25/55; 45.5%). Conclusion: Our results indicate that the 16S rRNA gene is more suitable for diagnosis of M. pneumoniae infection than the P1 adhesin gene due to its higher sensitivity and positive rate in clinical samples.

Clover proliferation phytoplasma: ‘Candidatus Phytoplasma trifolii’

2004 ◽  
Vol 54 (4) ◽  
pp. 1349-1353 ◽  
Author(s):  
Chuji Hiruki ◽  
Keri Wang
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Reference Strain ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Beet Leafhopper ◽  
Signature Sequences ◽  
Elm Yellows ◽  
The 16S Rrna Gene

Clover proliferation phytoplasma (CPR) is designated as the reference strain for the CP phylogenetic group or subclade, on the basis of molecular analyses of genomic DNA, the 16S rRNA gene and the 16S–23S spacer region. Other strains related to CPR include alfalfa witches'-broom (AWB), brinjal little leaf (BLL), beet leafhopper-transmitted virescence (BLTV), Illinois elm yellows (ILEY), potato witches'-broom (PWB), potato yellows (PY), tomato big bud in California (TBBc) and phytoplasmas from Fragaria multicipita (FM). Phylogenetic analysis of the 16S rRNA gene sequences of BLL, CPR, FM and ILEY, together with sequences from 16 other phytoplasmas that belong to the ash yellows (AshY), jujube witches'-broom (JWB) and elm yellows (EY) groups that were available in GenBank, produced a tree on which these phytoplasmas clearly clustered as a discrete group. Three subgroups have been classified on the basis of sequence homology and the collective RFLP patterns of amplified 16S rRNA genes. AWB, BLTV, PWB and TBBc are assigned to taxonomic subgroup CP-A, FM belongs to subgroup CP-B and BLL and ILEY are assigned to subgroup CP-C. Genetic heterogeneity between different isolates of AWB, CPR and PWB has been observed from heteroduplex mobility assay analysis of amplified 16S rRNA genes and the 16S–23S spacer region. Two unique signature sequences that can be utilized to distinguish the CP group from others were present. On the basis of unique properties of the DNA from clover proliferation phytoplasma, the name ‘Candidatus Phytoplasma trifolii’ is proposed for the CP group.

scholarly journals Reassessment of the phylogenetic relationships of Thiomonas cuprina

2007 ◽  
Vol 57 (11) ◽  
pp. 2720-2724 ◽  
Author(s):  
Donovan P. Kelly ◽  
Yoshihito Uchino ◽  
Harald Huber ◽  
Ricardo Amils ◽  
Ann P. Wood
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Significant Similarity ◽  
23S Rrna Gene ◽  
The 16S Rrna Gene

The published sequence of the 16S rRNA gene of Thiomonas cuprina strain Hö5 (=DSM 5495T) (GenBank accession no. U67162) was found to be erroneous. The 16S rRNA genes from the type strain held by the DSMZ since 1990 (DSM 5495T =NBRC 102145T) and strain Hö5 maintained frozen in the Universität Regensburg for 23 years (=NBRC 102094) were sequenced and found to be identical, but to show no significant similarity to the U67162 sequence. This also casts some doubt on the previously published 5S and 23S rRNA gene sequences (GenBank accession nos U67171 and X75567). The correct 16S rRNA gene sequence showed 99.8 % identity to those from Thiomonas delicata NBRC 14566T and ‘Thiomonas arsenivorans’ DSM 16361. The properties of these three species are re-evaluated, and emended descriptions are provided for the genus Thiomonas and the species Thiomonas cuprina.

scholarly journals Horizontal Transfer of Segments of the 16S rRNA Genesbetween Species of the Streptococcus anginosusGroup

2003 ◽  
Vol 185 (24) ◽  
pp. 7241-7246 ◽  
Author(s):  
Leo M. Schouls ◽  
Corrie S. Schot ◽  
Jan A. Jacobs
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Horizontal Transfer ◽  
Blot Hybridization ◽  
Reverse Line Blot ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Streptococcus Anginosus ◽  
The 16S Rrna Gene

ABSTRACT The nature in variation of the 16S rRNA gene of members of the Streptococcus anginosus group was investigated by hybridization and DNA sequencing. A collection of 708 strains was analyzed by reverse line blot hybridization. This revealed the presence of distinct reaction patterns representing 11 different hybridization groups. The 16S rRNA genes of two strains of each hybridization group were sequenced to near-completion, and the sequence data confirmed the reverse line blot hybridization results. Closer inspection of the sequences revealed mosaic-like structures, strongly suggesting horizontal transfer of segments of the 16S rRNA gene between different species belonging to the Streptococcus anginosus group. Southern blot hybridization further showed that within a single strain all copies of the 16S rRNA gene had the same composition, indicating that the apparent mosaic structures were not PCR-induced artifacts. These findings indicate that the highly conserved rRNA genes are also subject to recombination and that these events may be fixed in the population. Such recombination may lead to the construction of incorrect phylogenetic trees based on the 16S rRNA genes.

scholarly journals Comparative Genetic Diversity of the narG, nosZ, and 16S rRNA Genes in Fluorescent Pseudomonads

2003 ◽  
Vol 69 (2) ◽  
pp. 1004-1012 ◽  
Author(s):  
Sandrine Delorme ◽  
Laurent Philippot ◽  
Veronique Edel-Hermann ◽  
Chrystel Deulvot ◽  
Christophe Mougel ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pairwise Comparison ◽  
Fluorescent Pseudomonads ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Good Correspondence ◽  
The 16S Rrna Gene

ABSTRACT The diversity of the membrane-bound nitrate reductase (narG) and nitrous oxide reductase (nosZ) genes in fluorescent pseudomonads isolated from soil and rhizosphere environments was characterized together with that of the 16S rRNA gene by a PCR-restriction fragment length polymorphism assay. Fragments of 1,008 bp and 1,433 bp were amplified via PCR with primers specific for the narG and nosZ genes, respectively. The presence of the narG and nosZ genes in the bacterial strains was confirmed by hybridization of the genomic DNA and the PCR products with the corresponding probes. The ability of the strains to either reduce nitrate or totally dissimilate nitrogen was assessed. Overall, there was a good correspondence between the reductase activities and the presence of the corresponding genes. Distribution in the different ribotypes of strains harboring both the narG and nosZ genes and of strains missing both genes suggests that these two groups of strains had different evolutionary histories. Both dissimilatory genes showed high polymorphism, with similarity indexes (Jaccard) of between 0.04 and 0.8, whereas those of the 16S rRNA gene only varied from 0.77 to 0.99. No correlation between the similarity indexes of 16S rRNA and dissimilatory genes was seen, suggesting that the evolution rates of ribosomal and functional genes differ. Pairwise comparison of similarity indexes of the narG and nosZ genes led to the delineation of two types of strains. Within the first type, the similarity indexes of both genes varied in the same range, suggesting that these two genes have followed a similar evolution. Within the second type of strain, the range of variations was higher for the nosZ than for the narG gene, suggesting that these genes have had a different evolutionary rate.

scholarly journals Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline

2002 ◽  
Vol 184 (8) ◽  
pp. 2131-2140 ◽  
Author(s):  
Catharine A. Trieber ◽  
Diane E. Taylor
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Tetracycline Resistance ◽  
Clinical Isolates ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Nucleotide Substitutions ◽  
The 16S Rrna Gene

ABSTRACT Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 μg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H. pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 μg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H. pylori.

scholarly journals Pyrosequencing ofmcrAand Archaeal 16S rRNA Genes Reveals Diversity and Substrate Preferences of Methanogen Communities in Anaerobic Digesters

2014 ◽  
Vol 81 (2) ◽  
pp. 604-613 ◽  
Author(s):  
David Wilkins ◽  
Xiao-Ying Lu ◽  
Zhiyong Shen ◽  
Jiapeng Chen ◽  
Patrick K. H. Lee
Keyword(s):  
Anaerobic Digestion ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Methanogenic Archaea ◽  
16S Rrna Gene Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
The 16S Rrna Gene

ABSTRACTMethanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA) and 16S rRNA genes.Methanobacteriales,Methanomicrobiales, andMethanosarcinaleswere detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of themcrAgenes suggested that these digesters were dominated by acetoclastic methanogens, particularlyMethanosarcinales, while the other digesters were dominated by hydrogenotrophicMethanomicrobiales. The proposed euryarchaeotal orderMethanomassiliicoccalesand the uncultured WSA2 group were detected with the 16S rRNA gene, and potentialmcrAgenes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using themcrAgene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance ofmcrAtranscripts in digesters treating sludge and wastewater samples, supporting themcrAgene as a biomarker for methane yield.

scholarly journals Taxonomic note: speciation within the operational group Bacillus amyloliquefaciens based on comparative phylogenies of housekeeping genes

2020 ◽  
pp. 19-26
Author(s):  
Mohamad Syazwan Ngalimat ◽  
Suriana Sabri
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Analyses ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ratio Test ◽  
Group B ◽  
The 16S Rrna Gene ◽  
Operational Group

Many of the publically available Bacillus 16S rRNA genes and genomes in the NCBI database are inconsistently assigned as B. amyloliquefaciens. The highly conserved nature of the 16S rRNA gene makes it fail to differentiate species within the operational group B. amyloliquefaciens. Here, comparative phylogenies of the complete 16S rRNA, gyrB, rpoB, trpB, recA, and cheA nucleotide sequences of bacterial strains within the operational group were analyzed. As the result, the gyrB, rpoB, and trpB phylogenetic analyses showed stable topology that comprised three monophyletic clades: (i) B. amyloliquefaciens; (ii) B. siamensis; and (iii) B. velezensis. Phylogenies derived by comparison of the gyrB, rpoB, trpB, recA, and cheA with the 16S rRNA gene-derived phylogeny was significant as evaluated by the likelihood ratio test. The trpB, rpoB, and trpB gene-derived phylogenies provide a tool for speciation within the operational group B. amyloliquefaciens.

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 Detection and Identification of Banana-associated Phytoplasma Using Nested-PCR Method

2019 ◽  
Vol 23 (1) ◽  
pp. 148
Author(s):  
Saurma Mona Astrid Sibarani ◽  
Tri Joko ◽  
Siti Subandiyah
Keyword(s):  
16S Rrna ◽  
Nested Pcr ◽  
Plant Diseases ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Aster Yellows ◽  
Detection And Identification ◽  
Pcr Method ◽  
The 16S Rrna Gene

Phytoplasma is known to be associated with plant diseases in about 300 plant species from various families. Information on the presence of phytoplasma in bananas as one of the pathogens that can cause disease in bananas in Indonesia has never been reported. This research was conducted with the aim to detect the presence of banana phytoplasma by the nested-PCR method and to identify phytoplasma obtained based on the sequence analysis of the 16S rRNA gene. Standard PCR was carried out using P1/P7 primary pairs, followed by nested-PCR using a pair of R16F2n/R16R2m23SR primers separately that could amplify the target 16S rRNA genes in a row at 1600 bp. BLAST analysis shows that the results of phylogenetic analysis of banana phytoplasmic nucleotide cv. manggala from Tasikmalaya and cv. Raja nangka from Banjar has a genetic relationship that is closer to lethal wilt oil palm Phytoplasma (Candidatus Phytoplasma asteris). This phytoplasma belongs to the 16SrI-B group (aster yellows).

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