scholarly journals 16S rRNA-targeted polymerase chain reaction and oligonucleotide hybridization to screen for Azoarcus spp., grass-associated diazotrophs.

1993 ◽  
Vol 59 (11) ◽  
pp. 3816-3824 ◽  
Author(s):  
T Hurek ◽  
S Burggraf ◽  
C R Woese ◽  
B Reinhold-Hurek
Keyword(s):  
Polymerase Chain Reaction ◽  
16S Rrna ◽  
Chain Reaction ◽  
Oligonucleotide Hybridization ◽  
Polymerase Chain

scholarly journals Quantification of methanogenic Archaea within Baltic Sea copepod faecal pellets

2020 ◽  
Vol 167 (10) ◽  
Author(s):  
Janine Wäge ◽  
Oliver Schmale ◽  
Matthias Labrenz
Keyword(s):  
Polymerase Chain Reaction ◽  
16S Rrna ◽  
Baltic Sea ◽  
Methanogenic Archaea ◽  
Chain Reaction ◽  
Methanogenic Activity ◽  
Faecal Pellets ◽  
Analogous Data ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Abstract Methane enrichments are frequently observed in the oxic upper water column of the central Baltic Sea during summer months. However, methane sources as well as the fate of methane produced in surface near waters still remain unclear. In the present study, we conducted ship-based grazing experiments to examine the presence of methanogenic archaea in copepod faecal pellets. We quantified bacterial and archaeal 16S rRNA and the mcrA gene and transcripts within copepod faecal pellets by using droplet digital polymerase chain reaction. We showed that the pellets (< 150-µm) harbour a small number of methanogenic archaea; however, mcrA transcripts indicating methanogenic activity were not detected. This suggests that copepod faecal pellets from the central Baltic Sea, similar to analogous data on copepod guts, harbour the potential but are an unlikely hotspot for methane production by methanogenic archaea.

scholarly journals 1834. Incremental Diagnostic Value of 16S Ribosomal RNA Gene Polymerase Chain Reaction/Sanger Sequencing in Clinical Practice

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S44-S44
Author(s):  
Sarwat Khalil ◽  
Madiha Fida ◽  
Douglas W Challener ◽  
Omar Abu Saleh ◽  
Muhammad R Sohail ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Clinical Practice ◽  
16S Rrna ◽  
Ribosomal Rna ◽  
Mayo Clinic ◽  
Sanger Sequencing ◽  
Diagnostic Value ◽  
Chain Reaction ◽  
Material Support ◽  
Polymerase Chain

Abstract Background Polymerase chain reaction (PCR)/sequencing targeting the 16S ribosomal RNA (rRNA) gene to detect bacteria in normally sterile tissues and fluids has become increasingly popular in clinical medicine. This culture-independent technique can detect bacteria that are nonviable or difficult to cultivate using conventional methods. The clinical value of this type of testing is not well defined. We aimed to assess the diagnostic value of 16S rRNA PCR/Sanger sequencing as a clinical diagnostic assay at Mayo Clinic. Methods This is an interim analysis of the first 173 of 478 patients who had 16S rRNA PCR/Sanger sequencing done on sterile tissues or fluids at our institution from April, 2017 to November, 2018 as part of routine clinical practice. Medical records are being retrospectively reviewed, with results compared with those of culture. Results We reviewed 207 specimens from 173 patients (musculoskeletal 79%, cardiovascular 7%, central nervous system 4%, other 9%) that underwent 16S rRNA PCR/Sanger sequencing by clinical request (Table 1). In 90% of these specimens, the test was pre-planned rather than added-on. Nine specimens were excluded from analysis, as cultures were not performed. Overall concordance of culture with PCR/sequencing was 81% (160/197; P < 0.0001). Of 44 culture-positive specimens, PCR detected the same bacterium in 21 (48%) (Table 2). 45% (20/44) of those with positive cultures and 46% of those with positive PCR/sequencing results had received prior antimicrobial therapy (Table 3). PCR was negative in 139/144 specimens that were culture-negative (97%). PCR/sequencing was helpful in detecting a putative bacterial pathogen in 4 patients with negative cultures (Table 4). Conclusion Overall, 16S rRNA PCR/Sanger sequencing improved diagnostic yield compared with culture in a minority of cases. The described assay is limited by its inability to detect polymicrobial infections, a technical limitation that could possibly be addressed using massive parallel sequencing. Careful selection of cases and a save and add-on approach may be more cost-effective than upfront testing, although this was requested in a minority of cases. Disclosures Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

Polymerase Chain Reaction for the Rapid Detection of Cerebrospinal Fluid Shunt or Ventriculostomy Infections

Neurosurgery ◽  
2005 ◽  
Vol 57 (6) ◽  
pp. 1237-1243 ◽  
Author(s):  
Jason T. Banks ◽  
Suman Bharara ◽  
R Shane Tubbs ◽  
Charles L. Wolff ◽  
G Yancey Gillespie ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Polymerase Chain Reaction ◽  
Cerebrospinal Fluid ◽  
16S Rrna ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Propionibacterium Acnes ◽  
Chain Reaction ◽  
Gram Negative ◽  
Methicillin Resistant ◽  
Polymerase Chain

AbstractOBJECTIVE:Infection after cerebrospinal fluid (CSF) shunts or ventriculostomies is a common complication associated with significant morbidity and mortality. Polymerase chain reaction (PCR) is a powerful molecular technique that allows rapid and precise amplification of bacterial deoxyribonucleic acid (DNA) and has proven a powerful tool in the detection of a wide variety of clinically important infectious diseases. We analyzed specimens of CSF derived from ventriculoperitoneal shunts or external ventricular drains by using both conventional cultures and PCR and report herein our preliminary results.METHODS:We selected 86 CSF samples from adult patients who underwent either shunt tap or routine surveillance cultures of their ventriculostomy. These specimens were chosen from a larger group of 300 specimens that were routinely collected (many serially) in our clinical practice. They were chosen because clinical suspicion of infection was increased because of either patient signs and symptoms (fever, stiff neck, lethargy, worsening neurological examination) or preliminary laboratory analysis of CSF data (increased white blood cell count, increased protein level, decreased glucose). We considered this subgroup optimal to efficiently initiate our investigation of the correlation of PCR and culture results. CSF was increased by using standard culture techniques and by using PCR. Samples of CSF that were to undergo PCR had DNA extracted, purified, and amplified for 16S rRNA using primers 16S-Forward and 16S-Reverse of conserved sequence regions of all bacteria. DNA was PCR-amplified for 30 cycles. One microliter of the first PCR product was subjected to nested PCR using primers specific for gram-positive and gram-negative bacteria. Samples were also subjected to PCR amplification for specific detection of Propionibacterium acnes, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus using specific primers for 16S rRNA Propionibacterium, nuclease gene of Staphylococcus, and Mec gene of methicillin-resistant Staphylococcus aureus.RESULTS:For 18 of 86 specimens (21%), both the culture and PCR were positive. For 30 of 86 specimens (35%), both the PCR and culture results were negative. For 42 of 86 specimens (49%), cultures were negative and PCR was positive. There were no positive culture results with negative PCR results. Most negative culture/positive PCR cases occurred after prolonged intravenous antibiotics. Of the 56 PCR-positive specimens, 30 were positive for Propionibacterium acnes, whereas 40 were positive for Staphylococcus aureus. Of the Staphylococcus aureus-positive specimens, two were positive for methicillin resistant-Staphylococcus aureus. Among the 56 PCR-positive specimens, 30 were positive for both Propionibacterium acnes and Staphylococcus aureus; gram-negative organisms were not detected by any method in these specimens.CONCLUSION:These preliminary data suggest that PCR is a highly sensitive, rapid, and potentially promising modality for the detection and treatment of CSF shunt ventriculostomy infection.

Phenotypic and genotypic analysis of Flavobacterium psychrophilum isolates from Ontario salmonids with bacterial coldwater disease

2008 ◽  
Vol 54 (8) ◽  
pp. 619-629 ◽  
Author(s):  
Shohreh Hesami ◽  
Katie J. Allen ◽  
Devon Metcalf ◽  
Vaughn E. Ostland ◽  
Janet I. MacInnes ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Rainbow Trout ◽  
16S Rrna ◽  
Sequence Type ◽  
Chain Reaction ◽  
Flavobacterium Psychrophilum ◽  
Genotypic Analysis ◽  
Polymerase Chain ◽  
Coldwater Disease ◽  
Bacterial Coldwater Disease

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome. BCWD has a considerable economic impact on aquaculture operations in Ontario, Canada, and our limited understanding of the population structure and epidemiology of F. psychrophilum isolates is an impediment to the development of improved management strategies. Seventy-five 16S rRNA gene and gyr polymerase chain reaction positive isolates of F. psychrophilum that had been collected over a 16-year period from farmed salmonids with tail rot, necrotic myositis, and osteochondrosis were characterized morphologically, biochemically, and genotypically. Although the isolates were homogeneous by preliminary biochemical and phenotypic characterization, two distinct biovars were found by API ZYM testing. As well, four restriction pattern types were detected by 16S rRNA polymerase chain reaction – restriction fragment length polymorphism analysis and there was a significant (P < 0.001) correlation between biovar I and digestion with MaeIII and between biovar II and digestion with MnlI or no site (P < 0.05). Further heterogenity was detected by sequence analysis of a 194 bp stem loop 3 region of rRNA. Nine sequence types were identified; 40/46 biovar I isolates were sequence type “a”, while 21/32 biovar II isolates belonged to either sequence type “c” or “d”. More than one biovar and genotype was identified among the strains recovered from separate fish sampled from three groups of rainbow trout ( Oncorhynchus mykiss ) experiencing BCWD mortality events. No association was found between genotype or biovar and type of disease. Taken together, these data suggest that F. psychrophilum from Ontario can be grouped into two major lineages based on biovar and 16S rRNA polymorphisms, and although three major strain types were most frequently isolated in this study, it appears that the population of F. psychrophilum with pathogenic potential is quite heterogeneous.

Sign in / Sign up

Export Citation Format

Share Document