scholarly journals Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR Inhibitor Sodium Polyanetholesulfonate

1998 ◽  
Vol 36 (10) ◽  
pp. 2810-2816 ◽  
Author(s):  
David N. Fredricks ◽  
David A. Relman
Keyword(s):  
Blood Culture ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Benzyl Alcohol ◽  
Culture Media ◽  
Technical Problem ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Pcr Inhibitor ◽  
Purification Methods

Molecular methods are increasingly used to identify microbes in clinical samples. A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors. Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason. The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media. Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol. Accordingly, SPS tends to copurify with DNA. An extraction method was designed for purification of DNA from blood culture media and removal of SPS. Blood culture media containing human blood and spiked with Escherichia coli was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically. Successful amplification of the extracted E. coli 16S rRNA gene was achieved by adding 5 μl of undiluted processed sample DNA to a 50-μl PCR mixture. When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples. By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR. We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance.

scholarly journals 16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles

Microbiome ◽  
2014 ◽  
Vol 2 (1) ◽  
pp. 31 ◽  
Author(s):  
Jun Hang ◽  
Valmik Desai ◽  
Nela Zavaljevski ◽  
Yu Yang ◽  
Xiaoxu Lin ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Temperature Stability ◽  
Clinical Samples ◽  
Rrna Gene ◽  
16S Rrna Gene Pyrosequencing

scholarly journals Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

2002 ◽  
Vol 68 (10) ◽  
pp. 5064-5081 ◽  
Author(s):  
Alexander Loy ◽  
Angelika Lehner ◽  
Natuschka Lee ◽  
Justyna Adamczyk ◽  
Harald Meier ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Amplification ◽  
Oligonucleotide Microarray ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Sulfate Reducing ◽  
Specific Pcr

ABSTRACT For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

scholarly journals Streptococcus rupicaprae sp. nov., isolated from a Pyrenean chamois (Rupicapra pyrenaica)

2011 ◽  
Vol 61 (8) ◽  
pp. 1989-1993 ◽  
Author(s):  
A. I. Vela ◽  
G. Mentaberre ◽  
I. Marco ◽  
R. Velarde ◽  
S. Lavín ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequence ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Biochemical Tests ◽  
Pyrenean Chamois

Biochemical and molecular genetic studies were performed on an unknown Gram-stain-positive, catalase-negative, coccus-shaped organism isolated from clinical samples of a Pyrenean chamois. The micro-organism was identified as a streptococcal species based on its cellular morphological and biochemical tests. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus, but the organism did not correspond to any species of this genus. The nearest phylogenetic relative of the unknown coccus from chamois was Streptococcus ovis (95.9 % 16S rRNA gene sequence similarity). The rpoB and sodA sequence analysis showed sequence similarity values of less than 85.7 % and 83.0 %, respectively, with the currently recognized species of the genus Streptococcus. The novel bacterial isolate was distinguished from S. ovis and other species of the genus Streptococcus using biochemical tests. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a novel species of the genus Streptococcus, Streptococcus rupicaprae sp. nov., with the type strain 2777-2-07T ( = CECT 7718T  = CCUG 59652T).

scholarly journals Screening of Actinomycetes from Turmeric (Curcuma longa L.) and Ginger (Zingiber officinale) Rhizosphere for Antifungal Activity

2020 ◽  
pp. 18-28
Author(s):  
R. C. Osaro-Matthew ◽  
F. S. Ire ◽  
N. Frank-Peterside
Keyword(s):  
Antifungal Activity ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Culture Media ◽  
Curcuma Longa ◽  
Zingiber Officinale ◽  
Culture Technique ◽  
Rrna Gene ◽  
Dual Culture ◽  
Test Organisms

Aim: The present study was carried out to isolate actinomycetes from rhizospheric soil of Curcuma longa and Zingiber officinale and evaluate their antifungal potential. Methods: Actinomycetes were isolated from the rhizosphere soil of two Zingiberaceae plants (Curcuma longa and Zingiber officinale), using four different culture media. Isolates were screened for antifungal activity using dual culture technique against two reference strains Colletotrichum coccodes (DSM 2492) and Alternaria pimpriana (DSM 62023). The most potent strain was identified based on 16S rRNA gene sequence and the bioactive components of the strain were identified using GC-MS chromatography. Results: Fifteen strains of actinomycetes were isolated, SCA (starch casein agar) was found best for cultivation of actinomycetes. The 15 strains were grouped into three genera Norcadia 8(54%), Streptomyces 5(33%) and Janibacter 2(13%) based on morphological, biochemical and physiological identification. Among the 15 isolates 6(40%) strains showed activity against either of the test organisms, while 1(6.7%) was active against the both test organisms. Comparative analysis of the 16S rRNA gene sequences identified the potent isolates as Janibacter sp. strain RC18. GC-MS analysis revealed the presence of 20 compounds with 10 identified as potent antimicrobial metabolites. Conclusion: This study has revealed that rhizosphere of ginger and turmeric harbours rare actinomycetes that are valuable tool for sustainable agriculture.

scholarly journals Phylogenetic tree analysis based on the 16S sequence alignment for Klebsiella spp. isolated from different sources

2019 ◽  
pp. 1957-1966
Author(s):  
Mustafa Basil Abdul Qader ◽  
Marwa Hameed AlKhafaji
Keyword(s):  
16S Rrna ◽  
Klebsiella Pneumoniae ◽  
16S Rrna Gene ◽  
Enteric Bacteria ◽  
Apium Graveolens ◽  
Clinical Samples ◽  
Identification System ◽  
Rrna Gene ◽  
Vitek 2 ◽  
Klebsiella Spp

16S ribosomal RNA (16S rRNA) gene sequences used to study bacterial phylogeny and taxonomy have been by far the most common housekeeping genetic marker utilized for identification and ancestor determination. This study aimed to investigate, for the first time, the relationship between Klebsiella spp. isolated from clinical and environmental samples in Iraq.      Fifty Klebsiella spp. isolates were isolated from clinical and environmental sources. Twenty-five isolates were collected from a fresh vegetable (Apium graveolens) and 25 from clinical samples (sputum, wound swab, urine). Enteric bacteria were isolated on selective and differential media and identified by an automatic identification system, vitek-2. The total DNA was extracted and PCR amplified for selected isolates. The 16S rRNA gene was amplified by using the universal primer 27F (5'- AGAGTTTGATCCTGGCTCAG- 3') and 1492R (5'- GGTTACCTTGTTACGACTT- 3’). The 16SrRNA gene sequence was analysed among some local isolates, and the results were compared with the standard data of similar registered strains in NCBI. The most common species of Klebsiella was Klebsiella pneumoniae pneumoniae (Kpp), followed by Klebsiella pneumoniae ozaenae (Kpo) and Klebsiella oxytoca (Ko). The results of the identification of species and sub species by using the  biochemical test (vitek-2) were more precise than those obtained by the use of the universal primer.Phylogenetic tree strategies have clearly indicated a relatively close similarity amongst all analysed Klebsiella isolates and revealed the intra-species genetic distance between the individual isolates of the Klebsiella spp. In conclusion, our results revealed the main advantage of using universal primers for the identification of Klebsiella spp. and their root from nature.

scholarly journals Impact of DNA purification method and primer selection on 16S rRNA gene metabarcoding on wine

OENO One ◽  
2019 ◽  
Vol 53 (3) ◽  
Author(s):  
Francesco Cerutti ◽  
Diego Cravero ◽  
Antonella Costantini ◽  
Laura Pulcini ◽  
Paola Modesto ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Alternative Methods ◽  
Dna Purification ◽  
Rrna Gene ◽  
Purification Method ◽  
Main Species ◽  
Purification Methods ◽  
Primer Sets ◽  
The 16S Rrna Gene

Aim: The high-throughput sequencing methods have revolutionized the study of the microbiota in different matrices including those of the grapevine production chain. DNA extraction is a crucial step in the sample processing. In this study, we compared different DNA purification methods and two primer sets for 16S rRNA gene metabarcoding to evaluate the best protocol to explore the wine microbiota by metabarcoding.Methods and results: We collected a wine from Barbera grapes after malolactic fermentation previously inoculated by Oenococcus oeni starter. The same sample was used to evaluate the best performing protocol to study the wine microbiota. DNA was purified using nine different methods and then amplified for the 16S rRNA gene with two primer sets (according to Illumina or Earth Microbiome Project protocols). The obtained amplicons were then sequenced in a single sequencing session on an Illumina MiSeq. We evaluated the best protocol considering DNA concentration and purity, alpha (Observed species) and beta diversity from metabarcoding analysis.The sequencing generated 36,031,756 reads in total. Although no statistically significant difference was observed between purification methods or primer sets, better results were obtained with phenol-chloroform DNA purification combined to Earth Microbiome Project primers.Metabarcoding was able to highlight the domination of the inoculum, O. oeni, representing the main species of the analyzed wine microbiota.Conclusion: Our data show that, for the tested wine, metabarcoding output is more influenced by the primer set than by the DNA purification method. Moreover, the metabarcoding detected that O. oeni represents the main species, evidencing the domination of the inoculum done with lyophilized commercial preparation of this species. Other lactic acid bacteria are present at a much lower abundance.Significance and impact of the study: This is the first report applying the 16S rRNA gene metabarcoding to study the microbiota of wine. For this reason, the evaluation of alternative methods for DNA processing is essential for future research using this innovative methodology.

scholarly journals Ochrobactrum pseudintermedium sp. nov., a novel member of the family Brucellaceae, isolated from human clinical samples

2007 ◽  
Vol 57 (5) ◽  
pp. 1007-1013 ◽  
Author(s):  
Corinne Teyssier ◽  
Hélène Marchandin ◽  
Hélène Jean-Pierre ◽  
Agnès Masnou ◽  
Ghislaine Dusart ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
Clinical Samples ◽  
Rrna Gene ◽  
The Family ◽  
Ochrobactrum Intermedium ◽  
Physiological And Biochemical ◽  
The 16S Rrna Gene

Three novel Gram-negative, non-fermenting aerobic bacilli were isolated from human clinical samples. They shared more than 99.8 % of the 16S rRNA gene nucleotide positions. The strains were related to Ochrobactrum intermedium with about 97.48 % 16S rRNA gene sequence similarity. In 16S rRNA gene-, dnaK- and rpoB-based phylogenies, the strains were grouped in a lineage that was distinct from other Ochrobactrum species in the family Brucellaceae. Fatty acid composition, polar lipids, quinone system, DNA–DNA relatedness, genome organization, and physiological and biochemical data differentiated these isolates from recognized species of the genus Ochrobactrum. The three clinical strains therefore represent a novel species within the genus Ochrobactrum, for which the name Ochrobactrum pseudintermedium sp. nov., is proposed. The type strain is ADV31T (=CIP 109116T=DSM 17490T). The DNA G+C content of strain ADV31T was 54.5 mol%.

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