Bacterial Etiology for Chronic Villitis is Not Supported by Polymerase Chain Reaction for 16S rRNA DNA

2005 ◽  
Vol 8 (6) ◽  
pp. 647-653 ◽  
Author(s):  
Linda M. Ernst ◽  
Jill Crouch ◽  
Henry Rinder ◽  
John Greg Howe
Keyword(s):  
Polymerase Chain Reaction ◽  
16S Rrna ◽  
Plasma Cells ◽  
Infectious Agent ◽  
Pcr Primers ◽  
Proteinase K ◽  
Chain Reaction ◽  
Direct Spread ◽  
Polymerase Chain ◽  
Chronic Villitis

Chronic villitis is characterized by chorionic villi infiltrated by lymphocytes, histiocytes, and sometimes plasma cells. In a small percentage of cases, an infectious agent can be demonstrated within areas of chronic villitis. However, the pathogenesis of most lesions is idiopathic. Chronic villitis may represent the direct spread of chronic endometrial infection by bacterial organisms that are particularly problematic for culture. To test this hypothesis, polymerase chain reaction (PCR) using primers for the universal bacterial 16S rRNA DNA was performed on DNA extracted from areas of chronic villitis selected from placentas in the Yale Pathology database. Specific areas of chronic villitis were first confirmed by examination of sections stained with hematoxylin and eosin and then removed from archived paraffin blocks. Control tissue spiked with known bacterial counts was also prepared to test the sensitivity of the experiment. All tissue was deparaffinized, dehydrated, and digested with proteinase K. DNA extraction was performed with the Gentra Puregene kit. PCR was done using primers p11 and p13 for the 16S rRNA DNA. The 233-bp amplified target product was identified by agarose gel electrophoresis. Nineteen specimens with multifocal chronic villitis without confinement to anchoring villi were studied. None of the chronic villitis specimens had a demonstrable product using the PCR primers for 16S rRNA DNA, despite adequate DNA in the samples and controls. The assay was sensitive down to approximately 1500 bacteria per specimen. In conclusion, these data do not support a bacterial etiology for chronic villitis.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

Detection ofErwinia amylovorain plant material using novel polymerase chain reaction (PCR) primers

2001 ◽  
Vol 29 (1) ◽  
pp. 35-43 ◽  
Author(s):  
R. K. Taylor ◽  
P. J. Guilford ◽  
R. G. Clark ◽  
C. N. Hale ◽  
R. L. S. Forster
Keyword(s):  
Polymerase Chain Reaction ◽  
Plant Material ◽  
Pcr Primers ◽  
Chain Reaction ◽  
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.

Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

2006 ◽  
Vol 52 (5) ◽  
pp. 451-461 ◽  
Author(s):  
S S Hynes ◽  
O Chaudhry ◽  
M A Providenti ◽  
M L Smith
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Environmental Persistence ◽  
Fungal Strains ◽  
Target Dna ◽  
Polymerase Chain

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (~14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.Key words: Canada domestic substances list (DSL), Canadian Environmental Protection Act (CEPA), genetically modified organisms (GMO), quantitative polymerase chain reaction (qPCR).

Early Detection of Systemic Rust Infections of Dyers Woad (Isatis tinctoria) Using the Polymerase Chain Reaction

Weed Science ◽  
1995 ◽  
Vol 43 (3) ◽  
pp. 467-472 ◽  
Author(s):  
Bradley R. Kropp ◽  
Steve Albee ◽  
Karen M. Flint ◽  
Paul Zambino ◽  
Les Szabo ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Early Detection ◽  
Room Temperature ◽  
Detection Method ◽  
Rust Fungus ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Isatis Tinctoria ◽  
Specific Polymerase Chain Reaction ◽  
Polymerase Chain

Rust-specific polymerase chain reaction (PCR) primers selectively amplified ribosomal DNA of a rust fungus from infected dyers woad. PCR enabled DNA of the fungus to be detected in symptomatic plants as well as in asymptomatic parts of diseased plants. The use of PCR enabled early detection of rust infections in dyers woad plants during their first season when they are often asymptomatic Dried plant samples stored at room temperature for several months worked as well as lyophilized material for DNA extraction prior to PCR. The PCR detection method should greatly facilitate further studies on the biology and inoculation of this and other systemic rusts that have potential for use in biocontrol of weeds.

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