scholarly journals Optimization of denaturing high performance liquid chromatography technique for rapid detection and identification of acetic acid bacteria of interest in vinegar production

2013 ◽  
Vol 2 (1s) ◽  
pp. 5 ◽  
Author(s):  
Noelia Isabel Sagarzazu ◽  
Maribel Martínez ◽  
Cristina Algarra ◽  
Javier Butrón ◽  
Carlos J. González-Navarro ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Acetic Acid ◽  
Liquid Chromatography ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Performance ◽  
Acetic Acid Bacteria ◽  
Gene Region ◽  
Rrna Gene ◽  
Wild Strains

This paper evaluates the use of denaturing high performance liquid chromatography (DHPLC) technology for the discrimination of genetic differences in the 16S rRNA and alcohol dehydrogenase (AdhA) genes among bacterial species based on its efficiency and sensitivity to enable the detection and discrimination of different genetic sequences. In order to optimize DHPLC protocols for the analysis of 16S rRNA gene fragments amplified from bacteria, DNA isolated from 22 different strains representing main bacterial groups of interest in food microbiology was analyzed. While the use of 16S rRNA gene did not allow to difference two wild strains of <em>Acetobacter malorum</em>, this region revealed as useful to differentiate them from some pathogenic bacteria as <em>Escherichia coli</em>, <em>Salmonella typhimurium</em>, <em>Listeria monocytogenes</em>, <em>Listeria innocua</em>, <em>Clostridium perfringens </em>or <em>Sthapylococcus aureus</em>, from spoilage microorganisms as <em>Xantomonas vesicatoria</em> and <em>Alicyclobacillus</em> spp., and also from lactic acid bacteria as <em>Lactobacillus plantarum</em>, <em>Lactobacillus casei</em>, <em>Lactobacillus sakei</em>, <em>Lactobacillus acidophilus</em>, <em>Streptococcus thermophilus </em>and <em>Lactococcus lactis</em> that may suppose technological risk during vinegar production. The results demonstrate that 16S rRNA gene region is not adequate for the discrimination of the acetic acid bacteria (AAB) strains, so AdhA gene was selected to identify the two wild strains of <em>Acetobacter malorum</em>. Also 6 different reference strains of AAB were separated based on differences in AdhA gene region. DHPLC technology is able to discriminate between these two wild strains of <em>A. malorum</em> based on differences existing in the AdhA gene region. The data obtained indicate that the technique is capable of identifying most bacteria at species level and even at strain level with optimization of the protocols. This is of particular relevance in the case of AAB due to their poor recovery on culture media and difficulties in detection of viable but non cultivable cells.

scholarly journals Molecular identification of acetic acid bacteria isolated from fermented mango juices of Burkina Faso: 16S rRNA gene sequencing

2019 ◽  
Vol 18 (29) ◽  
pp. 766-773
Author(s):  
OUATTARA Assiètta ◽  
Marius SOMDA K. ◽  
T. Cheik OUATTARA A. ◽  
N’DOYE Bassirou ◽  
TRAORE Alfred ◽  
...  
Keyword(s):  
Acetic Acid ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Burkina Faso ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Acetic Acid Bacteria ◽  
Rrna Gene ◽  
Rrna Gene Sequencing ◽  
Mango Juices

scholarly journals Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Christine Drengenes ◽  
Tomas M. L. Eagan ◽  
Ingvild Haaland ◽  
Harald G. Wiker ◽  
Rune Nielsen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Load ◽  
Marker Gene ◽  
Gene Region ◽  
Lower Airway ◽  
Rrna Gene ◽  
Wide Range ◽  
Airway Microbiome ◽  
The Impact

Abstract Background Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. Results The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. Conclusions Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

scholarly journals Influence of Indole acetic acid and Tryptophan on production of Vinblastine and Vincristine of Catharanthus roseous callus cells in the accumulation media of In Vitro tissue culture

2010 ◽  
Vol 7 (3) ◽  
pp. 1113-1119
Author(s):  
Baghdad Science Journal
Keyword(s):  
Tissue Culture ◽  
High Performance Liquid Chromatography ◽  
Acetic Acid ◽  
Liquid Chromatography ◽  
In Vitro Culture ◽  
High Performance ◽  
Indole Acetic Acid ◽  
Culture Technique

This study on the plant of Ain –AL Bason Catharanthus roseous showed the ability of callus cells that is produced by In Vitro culture technique and transformed to the accumulated media (MS 40gm/L sucrose ,2gm/L IAA Indole acetic acid , 0.5gm/L Tryptophan) to produce Vinblastine and Vincristine compounds. Extraction, purification and quantitive determination of Vinblastine and Vincristine compounds using High performance liquid chromatography technique (HPLC)were carried out. The results showed that the highest concentration of Vinblastine and Vincristine compounds were ( 4.653,12.5 (ppm /0.5 dry Wight respectively from transformed callus cells from MS 40 gm /L sucrose , 2 gm / L NAA Naphthaline acetic acid .

scholarly journals Development of a Denaturing High-Performance Liquid Chromatography Method for Detection of Protist Parasites of Metazoans

2008 ◽  
Vol 74 (14) ◽  
pp. 4336-4345 ◽  
Author(s):  
Christofer Troedsson ◽  
Richard F. Lee ◽  
Vivica Stokes ◽  
Tina L. Walters ◽  
Paolo Simonelli ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Chromatography Method ◽  
Factors Affecting ◽  
Triethylammonium Acetate

ABSTRACT Increasingly, diseases of marine organisms are recognized as significant biotic factors affecting ecosystem health. However, the responsible disease agents are often unknown and the discovery and description of novel parasites most often rely on morphological descriptions made by highly trained specialists. Here, we describe a new approach for parasite discovery, utilizing denaturing high-performance liquid chromatography (DHPLC) reverse-phase ion-paring technology. Systematic investigations of major DHPLC variables, including temperature, gradient conditions, and target amplicon characteristics were conducted to develop a mechanistic understanding of DNA fragment separation by DHPLC. As a model system, 18S rRNA genes from the blue crab (Callinectes sapidus) and a parasitic dinoflagellate Hematodinium sp. were used. Binding of 18S rRNA gene PCR amplicons to the DNA separation column in the presence of triethylammonium acetate (TEAA) was inversely correlated with temperature and could be predicted based on the estimated DNA helicity of the PCR amplicon. Amplicons of up to 498 bp were resolved as single chromatographic peaks if they had high (>95%) DNA helicity. Amplicons that differed by as few as 2 bp could be resolved. Separation of 18S rRNA gene PCR amplicons was optimized by simultaneous manipulation of both temperature and solvent gradients. The optimal conditions included targeting regions of high DNA helicity (>95%), temperatures in the range of 57 to 63°C, and a linear acetonitrile gradient from 13.75 to 17.5% acetonitrile in 0.1 M TEAA (55 to 70% buffer B) over a 9-min period. Under these conditions, amplicons from a variety of parasites and their hosts can be separated and detected by DHPLC.

scholarly journals Gluconobacter nephelii sp. nov., an acetic acid bacterium in the class Alphaproteobacteria

2011 ◽  
Vol 61 (9) ◽  
pp. 2117-2122 ◽  
Author(s):  
Jintana Kommanee ◽  
Somboon Tanasupawat ◽  
Pattaraporn Yukphan ◽  
Taweesak Malimas ◽  
Yuki Muramatsu ◽  
...  
Keyword(s):  
Acetic Acid ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Trees ◽  
23S Rrna ◽  
Rrna Gene ◽  
Phenotypic Data ◽  
Dna Relatedness ◽  
23S Rrna Gene ◽  
Type Strains

Three strains, RBY-1T, PHD-1 and PHD-2, were isolated from fruits in Thailand. The strains were Gram-negative, aerobic rods with polar flagella, produced acetic acid from ethanol and did not oxidize acetate or lactate. In phylogenetic trees based on 16S rRNA gene sequences and 16S–23S rRNA gene internal transcribed spacer (ITS) sequences, the strains formed a cluster separate from the type strains of recognized species of the genus Gluconobacter. The calculated 16S rRNA gene sequence and 16S–23S rRNA gene ITS sequence similarities were respectively 97.7–99.7 % and 77.3–98.1 %. DNA G+C contents ranged from 57.2 to 57.6 mol%. The strains showed high DNA–DNA relatedness of 100 % to one another, but low DNA–DNA relatedness of 11–34 % to the tested type strains of recognized Gluconobacter species. Q-10 was the major quinone. On the basis of the genotypic and phenotypic data obtained, the three strains clearly represent a novel species, for which the name Gluconobacter nephelii sp. nov. is proposed. The type strain is RBY-1T ( = BCC 36733T = NBRC 106061T = PCU 318T), whose DNA G+C content is 57.2 mol%.

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