scholarly journals Bacterial DNA patterns identified using paired-end Illumina sequencing of 16S rRNA genes from whole blood samples of septic patients in the emergency room and intensive care unit

2018 ◽  
Vol 18 (1) ◽  
Author(s):  
Monica Martins Pereira Faria ◽  
Brent Warren Winston ◽  
Michael Gordon Surette ◽  
John Maynard Conly
Keyword(s):  
Intensive Care Unit ◽  
Intensive Care ◽  
16S Rrna ◽  
Emergency Room ◽  
Whole Blood ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Dna ◽  
Blood Samples ◽  
Whole Blood Samples

scholarly journals Diagnosis of Bacteremia in Whole-Blood Samples by Use of a Commercial Universal 16S rRNA Gene-Based PCR and Sequence Analysis

2009 ◽  
Vol 47 (9) ◽  
pp. 2759-2765 ◽  
Author(s):  
N. Wellinghausen ◽  
A.-J. Kochem ◽  
C. Disque ◽  
H. Muhl ◽  
S. Gebert ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Whole Blood ◽  
Rrna Gene ◽  
Blood Samples ◽  
Whole Blood Samples

scholarly journals Detection of Aspergillus fumigatus in Blood Samples from Critically Ill Patients in Intensive Care Units by Use of the SeptiFast Assay: TABLE 1

2016 ◽  
Vol 54 (7) ◽  
pp. 1918-1921 ◽  
Author(s):  
Joerg Steinmann ◽  
Jan Buer ◽  
Peter-Michael Rath
Keyword(s):  
Intensive Care Unit ◽  
Intensive Care ◽  
Positive Predictive Value ◽  
Aspergillus Fumigatus ◽  
Critically Ill ◽  
Predictive Value ◽  
Icu Patients ◽  
Blood Samples ◽  
Content Type ◽  
Whole Blood Samples

We retrospectively analyzed the performance and relevance of the SeptiFast assay in detectingAspergillus fumigatusDNA in whole blood samples from 38 critically ill intensive care unit (ICU) patients with probable or proven invasive aspergillosis (IA) and 100 ICU patients without IA. The assay exhibited 66% sensitivity, 98% specificity, a 93% positive predictive value, and an 88% negative predictive value.A. fumigatusDNAemia was associated with poor outcome.

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 Screening of a Fosmid Library of Marine Environmental Genomic DNA Fragments Reveals Four Clones Related to Members of the OrderPlanctomycetales

1998 ◽  
Vol 64 (8) ◽  
pp. 3075-3078 ◽  
Author(s):  
Kevin L. Vergin ◽  
Ena Urbach ◽  
Jeffery L. Stein ◽  
Edward F. DeLong ◽  
Brian D. Lanoil ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
Genomic Dna ◽  
Pcr Amplification ◽  
Fosmid Library ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacterial Dna ◽  
Marine Environmental

ABSTRACT A fosmid library with inserts containing approximately 40 kb of marine bacterial DNA (J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya, and E. F. DeLong, J. Bacteriol. 178:591–599, 1996) yielded four clones with 16S rRNA genes from the orderPlanctomycetales. Three of the clones belong to thePirellula group and one clone belongs to thePlanctomyces group, based on phylogenetic and signature nucleotide analyses of full-length 16S rRNA genes. Sequence analysis of the ends of the genes revealed a consistent mismatch in a widely used bacterium-specific 16S rRNA PCR amplification priming site (27F), which has also been reported in some thermophiles and spirochetes.

A technique for the measurement of orthophosphate in human erythrocytes, and some studies of its determinants

1985 ◽  
Vol 69 (4) ◽  
pp. 429-434 ◽  
Author(s):  
A. Challa ◽  
A. Bevington ◽  
C. M. Angier ◽  
A. J. Asbury ◽  
C. J. Preston ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Intensive Care ◽  
Whole Blood ◽  
Concentration Ratio ◽  
Normal Subjects ◽  
Human Erythrocytes ◽  
Blood Samples ◽  
Internal Standardization ◽  
Hydrolysis Of

1. A technique is described for the determination of orthophosphate (Pi) in human erythrocytes. The advantages of the technique are that it uses whole blood rather than separated erythrocytes, that it avoids major hydrolysis of organic phosphates, that it takes account of incomplete recovery of Pi and that it minimizes the effects of chilling the cells. 2. In chilled samples from 46 patients in an intensive care unit, the cellular concentration of Pi was proportional to that in plasma. 3. Blood samples from nine normal subjects were incubated at 37°C. The cellular Pi was 0.79 mmol/litre of cells using an external standardization and 0.67 using an internal standardization. When the same cell samples were chilled on ice for 30 min, the internally standardized value decreased further to 0.57 mmol/litre of cells. These results suggest that differences in recovery, and the extent of chilling, contribute to the variability in the previously reported values for erythrocyte Pi. 4. If Pi, like chloride, had distributed passively between cells and plasma in these samples, the cell to plasma molar concentration ratio for Pi should have been 0.29, compared with the measured value of 0.64. This difference suggests that some factor, in addition to passive diffusion, determined the distribution of Pi.

scholarly journals Contamination Is Not Linked to the Gestational Microbiome

2019 ◽  
Vol 85 (19) ◽  
Author(s):  
Michelle D. Rodriguez ◽  
Kevin K. Yu ◽  
Zubin S. Paul ◽  
Maureen Keller-Wood ◽  
Charles E. Wood ◽  
...  
Keyword(s):  
16S Rrna ◽  
Fetal Liver ◽  
In Utero ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Tissue Samples ◽  
Bacterial Dna ◽  
Culture Independent ◽  
Active Transfer ◽  
Colonization Hypothesis

ABSTRACT Differentiating between contamination and the genuine presence of 16S rRNA genes in gestational tissue samples is the gold standard for supporting the in utero colonization hypothesis. During gestation, the fetus undergoes significant physiological changes that may be directly affected by maternal colonization of key bacterial genera. In this study, lab benches, necropsy tables, and air ducts were swabbed at the same time as clinical sampling. The relative and absolute abundance of bacteria present in sheep samples was determined by culture-independent and culture-dependent means. Of 14 healthy pregnant ewes, there was no evidence of any bacteria in the fetal liver, spleen, or brain cortex using culture-independent techniques despite evidence of the presence of bacteria in various locations of the necropsy room used for 11 of these 14 sheep. Of the 336 bacterial genera found in the room swabs, only 12 (5%) were also found in the saliva and vaginal swabs among the three ewes for which bacteria were detected. These 12 taxa represent 1.32% of the relative abundance and approximately 393 16S rRNA copies/swab in these three ewes. Using careful necropsy protocols, bacterial contamination of sheep tissues was avoided. Contamination of saliva and vaginal samples was limited to less than 2% of the bacterial population. IMPORTANCE Recent evidence for a gestational microbiome suggests that active transfer between mother and fetus in utero is possible, and, therefore, actions must be taken to clarify the presence versus absence of these organisms in their respected sources. The value of this study is the differentiation between bacterial DNA identified in the necropsy rooms of animals and bacterial DNA whose origin is purely clinical in nature. We do not know the extent to which microorganisms traverse maternal tissues and infiltrate fetal circulation, so measures taken to control for contamination during sample processing are vital for addressing these concerns.

scholarly journals Elimination of Bacterial DNA from TaqDNA Polymerases by Restriction Endonuclease Digestion

1999 ◽  
Vol 37 (10) ◽  
pp. 3402-3404 ◽  
Author(s):  
Nora M. Carroll ◽  
Peter Adamson ◽  
Narciss Okhravi
Keyword(s):  
16S Rrna ◽  
Dna Polymerase ◽  
Restriction Enzyme ◽  
Clinical Applications ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Endonuclease Digestion ◽  
Bacterial Dna ◽  
Diagnosis Of Infection ◽  
Endonuclease Digestion

The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S rRNA genes. The method was used prior to a PCR that amplified 10 fg of bacterial DNA. This method can be readily adapted to suit other sensitive PCRs required for clinical applications.

Steadiness/stability of antiepileptic drugs in serum, plasma and whole-blood samples under various storage methods

2010 ◽  
Vol 41 (02) ◽  
Author(s):  
N Shazi ◽  
A Böss ◽  
HJ Merkel ◽  
F Scharbert ◽  
D Hannak ◽  
...  
Keyword(s):  
Antiepileptic Drugs ◽  
Whole Blood ◽  
Blood Samples ◽  
Storage Methods ◽  
Whole Blood Samples

Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

2015 ◽  
Vol 41 (1) ◽  
pp. 51-58
Author(s):  
Mohammad Shamimul Alam ◽  
Hawa Jahan ◽  
Rowshan Ara Begum ◽  
Reza M Shahjahan
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fish Larva ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Valuable Insight ◽  
Multiple Sequence ◽  
Pcr Rflp ◽  
Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

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