scholarly journals PCR Analysis of the Distribution of Unicellular Cyanobacterial Diazotrophs in the Arabian Sea

2004 ◽  
Vol 70 (12) ◽  
pp. 7355-7364 ◽  
Author(s):  
Sophie L. Mazard ◽  
Nicholas J. Fuller ◽  
Karen M. Orcutt ◽  
Oliver Bridle ◽  
Dave J. Scanlan
Keyword(s):  
Arabian Sea ◽  
Marine Diatom ◽  
Pcr Analysis ◽  
Oligonucleotide Primer ◽  
Rrna Gene ◽  
Key Factor ◽  
Subsurface Waters ◽  
Reverse Primer ◽  
The 16S Rrna Gene ◽  
Forward Primer

ABSTRACT An oligonucleotide primer, NITRO821R, targeting the 16S rRNA gene of unicellular cyanobacterial N2 fixers was developed based on newly derived sequences from Crocosphaera sp. strain WH 8501 and Cyanothece sp. strains WH 8902 and WH 8904 as well as several previously described sequences of Cyanothece sp. and sequences of intracellular cyanobacterial symbionts of the marine diatom Climacodium frauenfeldianum. This oligonucleotide is specific for the targeted organisms, which represent a well-defined phylogenetic lineage, and can detect as few as 50 cells in a standard PCR when it is used as a reverse primer together with the cyanobacterium- and plastid-specific forward primer CYA359F (U. Nübel, F. Garcia-Pichel, and G. Muyzer, Appl. Environ. Microbiol. 63:3327-3332, 1997). Use of this primer pair in the PCR allowed analysis of the distribution of marine unicellular cyanobacterial diazotrophs along a transect following the 67°E meridian from Victoria, Seychelles, to Muscat, Oman (0.5°S to 26°N) in the Arabian Sea. These organisms were found to be preferentially located in warm (>29°C) oligotrophic subsurface waters between 0 and 7°N, but they were also found at a station north of Oman at 26°N, 56°35′E, where similar water column conditions prevailed. Slightly cooler oligotrophic waters (<29°C) did not contain these organisms or the numbers were considerably reduced, suggesting that temperature is a key factor in dictating the abundance of this unicellular cyanobacterial diazotroph lineage in marine environments.

scholarly journals Demonstration of Bartonella grahamii DNA in Ocular Fluids of a Patient with Neuroretinitis

1999 ◽  
Vol 37 (12) ◽  
pp. 4034-4038 ◽  
Author(s):  
F. T. Kerkhoff ◽  
A. M. C. Bergmans ◽  
A. van der Zee ◽  
A. Rothova
Keyword(s):  
Gene Sequence ◽  
Dna Analysis ◽  
Behavioral Changes ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Pcr Product ◽  
Immunodeficiency Virus ◽  
Laboratory Features ◽  
The 16S Rrna Gene

We describe the clinical and laboratory features of a 55-year-old human immunodeficiency virus-negative female patient who presented with bilateral intraocular inflammatory disease (neuroretinitis type) and behavioral changes caused by a Bartonella grahamiiinfection. Diagnosis was based on the PCR analysis of DNA extracted from the intraocular fluids. DNA analysis of the PCR product revealed a 100% identity with the 16S rRNA gene sequence of B. grahamii. The patient was successfully treated with doxycycline (200 mg/day) and rifampin (600 mg/day) for 4 weeks. This is the first report that demonstrates the presence of a Bartonellaspecies in the intraocular fluids of a nonimmunocompromised patient and that indicates that B. grahamii is pathogenic for humans.

scholarly journals Intestinal Integrity and Akkermansia muciniphila, a Mucin-Degrading Member of the Intestinal Microbiota Present in Infants, Adults, and the Elderly

2007 ◽  
Vol 73 (23) ◽  
pp. 7767-7770 ◽  
Author(s):  
M. Carmen Collado ◽  
Muriel Derrien ◽  
Erika Isolauri ◽  
Willem M. de Vos ◽  
Seppo Salminen
Keyword(s):  
Early Life ◽  
Intestinal Tract ◽  
The Elderly ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Akkermansia Muciniphila ◽  
Intestinal Integrity ◽  
The 16S Rrna Gene ◽  
Human Intestinal Tract

ABSTRACT Fluorescence in situ hybridization and real-time PCR analysis targeting the 16S rRNA gene of Akkermansia muciniphila were performed to determine its presence in the human intestinal tract. These techniques revealed that an A. muciniphila-like bacterium is a common member of the human intestinal tract and that its colonization starts in early life and develops within a year to a level close to that observed in adults (108 cells/g) but decreases (P < 0.05) in the elderly.

scholarly journals Moleculer Detection of Protozoa Trichodina spp. In Gourami (Osphromenus Gourame Lac.) Larvae with The infecting 18S rRNA Gene Marking in Exs. Residence of Banyumas, Central Java

2018 ◽  
Vol 10 (2) ◽  
pp. 320-325
Author(s):  
Rokhmani Rokhmani ◽  
Endang Ariyani Setyawati ◽  
Daniel Joko Wahyono
Keyword(s):  
18S Rrna ◽  
18S Rrna Gene ◽  
Survey Method ◽  
Rrna Gene ◽  
Morphological Variations ◽  
Central Java ◽  
Reverse Primer ◽  
Partial 18S ◽  
Forward Primer

Protozoa species of Trichodina spp. may be found in most hatchery centers in Banyumas, Purbalingga, and Banjarnegara. However, the determination of Trichodina spp. types is still based on its body’s morphological variations, not yet molecular. A research has been conducted to identify molekuler of the Trichodina spp. with the infecting 18S rRNA gene marking on the gourami larvae in Exs. Residence of Banyumas, Central Java. The research used a survey method with the samples of gourami. Amplification of 18S rRNA gene from Trichodina heterodentata was Performed using PCR technique. Primer used is Forward primer (5 ‘-AAC CTG GTT GAT CCT GCC ATG-3’) and Reverse primer (5 ‘-TGA TCC TTC TGC AGG TTC ACC TAC-3’) which produces a 600 pb amplicon of DNA. Molecular research can be a complementary identification of organisms morphologically. Amplification of the partial 18S rRNA gene may be used to identify Trichodina specifically. Gourami larvae taken from the hatchery centers in Banyumas, Purbalingga, and Banjarnegara. The results show that the detected percentage of Trichodina heterodentata genes with the infecting 18S rRNA gene marking on the gourami larvae in Central Java taken from the hatchery centers in Banyumas, Purbalingga and Banjarnegara are respectively 10%, 10%, and 45%. This research provides a benefit in mapping the presence of protozoa pathogen of Trichodina spp. in gourami hatcheries in the Former Exs. Residence of Banyumas, Central Java

scholarly journals Analysis of 16S rRNA and 51-Kilodalton Antigen Gene and Transmission in Mice of Ehrlichia risticii in Virgulate Trematodes from Elimia livescens Snails in Ohio

2000 ◽  
Vol 38 (9) ◽  
pp. 3349-3358 ◽  
Author(s):  
Manuel Kanter ◽  
Jason Mott ◽  
Norio Ohashi ◽  
Bernard Fried ◽  
Stephen Reed ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Strain ◽  
Pcr Analysis ◽  
Composition Analysis ◽  
Rrna Gene ◽  
Antigen Gene ◽  
Ehrlichia Risticii ◽  
Horse Blood ◽  
The 16S Rrna Gene

Operculate snails (the family Pleuroceridae:Elimia livescens) were collected between June and October 1998 from a river in central Ohio where repeated cases of Potomac horse fever (PHF) have occurred. Of collected snails, consistently 50 to 80% carried a combination of cercariae and sporocysts of digenetic virgulate trematodes. The trematodes obtained from each snail were pooled and tested for Ehrlichia risticii, the agent of PHF, by nested PCR using primers specific to the 16S rRNA gene. Out of a total of 209 trematode pools, 50 pools were found to be positive by PCR. The DNA sequence of the 16S rRNA gene identified in one trematode pool was identical to that of the type strain of E. risticii, and the sequence of the gene identified in another pool differed from that of the type strain by 1 nucleotide. Comparison of the deduced amino acid sequence of the partial 51-kDa antigen gene from various sources revealed that Maryland, Ohio (except Ohio 081), and Kentucky strains are in a cluster distinct from the sequences obtained from sources in California and Oregon. Ohio 081 was shown previously by antigenic composition analysis to be distinct from other groups. However, all sequences examined were not segregated according to their sources: horse blood or infected trematodes. E. risticiiwas found to be transmittable from trematodes to mice and was subsequently passaged from infected mice to additional mice, as determined by PCR analysis. Our findings suggest the evolution ofE. risticii in the natural reservoir in separate geographic regions and persistent infection of trematode populations with E. risticii during summer and early fall. The study also suggests that the mouse can be used to isolate E. risticii from the infected trematode.

A coagulase-negative variant ofStaphylococcus aureusfrom bovine mastitis milk

2010 ◽  
Vol 78 (1) ◽  
pp. 38-42 ◽  
Author(s):  
Ömer Akineden ◽  
Abdulwahed Ahmed Hassan ◽  
Elisabeth Schneider ◽  
Ewald Usleber
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bovine Mastitis ◽  
Surface Protein ◽  
Negative Reaction ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Coagulase Negative Staphylococci ◽  
Staph Aureus ◽  
The 16S Rrna Gene

Bacteriological analysis of milk samples from quarters of a dairy cow suffering from subclinical mastitis yielded two isolates ofStaphylococcus aureuswhich gave a negative reaction in the standard coagulase test. Both isolates were also clumping factor and thermonuclease negative, and gave a negative reaction in the Staphaurex®test. The isolates were identified by using commercial biochemical systems, and by PCR analysis of different staphylococcal cell surface protein and exoprotein genes. Further molecular identification of the isolates, which included sequencing of the 16S rRNA gene and RT-PCR of coagulase (coa), clumping-factor (clfA) and thermonuclease (nuc) genes, was consistent with the diagnosis phenotypically ‘coagulase-negative variant ofStaph. aureus’. The fact that coagulase-negativeStaph. aureusvariants can occur in the context of intramammary infections in cattle may result in the incorrect diagnosis ‘coagulase-negative staphylococci (CNS)’ in routine mastitis diagnostic, at least in rare cases. To fully ensure correct species diagnosis, sequencing of the 16S rRNA gene and amplification of specific genes such ascoais necessary in these cases.

scholarly journals Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes

2008 ◽  
Vol 74 (8) ◽  
pp. 2461-2470 ◽  
Author(s):  
Jeremy A. Frank ◽  
Claudia I. Reich ◽  
Shobha Sharma ◽  
Jon S. Weisbaum ◽  
Brenda A. Wilson ◽  
...  
Keyword(s):  
16S Rrna ◽  
Critical Evaluation ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Amplification Efficiency ◽  
Rrna Gene ◽  
Minimal Loss ◽  
Reverse Primer ◽  
Pcr Conditions ◽  
Forward Primer

ABSTRACT rRNA-based studies, which have become the most common method for assessing microbial communities, rely upon faithful amplification of the corresponding genes from the original DNA sample. We report here an analysis and reevaluation of commonly used primers for amplifying the DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the Escherichia coli rRNA). We propose a formulation for a forward primer (27f) that includes three sequences not usually present. We compare our proposed formulation to two common alternatives by using linear amplification—providing an assessment that is independent of a reverse primer—and in combination with the 1492 reverse primer (1492r) under the PCR conditions appropriate for making community rRNA gene clone libraries. For analyses of DNA from human vaginal samples, our formulation was better at maintaining the original rRNA gene ratio of Lactobacillus spp. to Gardnerella spp., particularly under stringent amplification conditions. Because our 27f formulation remains relatively simple, having seven distinct primer sequences, there is minimal loss of overall amplification efficiency and specificity.

scholarly journals Investigation of the effects of some phytochemicals on Yersinia ruckeri and antimicrobial resistance

2020 ◽  
Vol 80 (4) ◽  
pp. 934-942
Author(s):  
Ş. Önalan ◽  
M. Çevik
Keyword(s):  
Moringa Oleifera ◽  
Biochemical Properties ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Bacterial Disease ◽  
Yersinia Ruckeri ◽  
Biochemical Tests ◽  
Positive Results ◽  
Day By Day ◽  
The 16S Rrna Gene

Abstract In this study, it is aimed to investigate the effects of Moringa oleifera and Sorbus domestica plant extracts on bacterial disease agents Yersinia ruckeri in aquaculture. Morphological and biochemical properties of 2 different Y. ruckeri isolates were determined. Then, Real-Time PCR analysis and gene sequencing of the isolates were identified. Phytochemicals (M. oleifera and S. domestica) and antibiotics (Oxytetracycline (OX) and Enrofloxacin (ENR)) were used together in the antibiogram test of antibiotics compared to the effect status of antibiotics. Also, the effects of phytochemicals on Y. ruckeri growth was examined comparatively by spectrophotometrically measuring at 600 nm wavelength every 2 hours according to bacterial growth densities with 10 different groups formed on TSB medium. As a result of the study, it was observed that the isolates formed Gram negative, catalase positive, oxidase negative, mobile and typical Y. ruckeri colonies. After the biochemical tests performed with Microgen ID panel, 99.85% similarity was determined. The isolates overlap with the 16S rRNA gene region after sequence analysis, and 99% of the isolates were similar in phylogenetic analysis. After the antibiogram test, Oxytetracycline and Enrofloxacin antibiotics were resistant to Y. ruckeri but the effects of phytochemicals were less on solid medium (MHA). As a result of the measurements carried out in liquid medium (TSB), it was observed that phytochemicals such as M. oliefera and S. domestica inhibit the growth of bacteria by 40-50%. As the importance of antibiotic resistance is increasing day by day, we believe that these phytochemicals will give positive results in treatment instead of using antibiotics.

scholarly journals Improvement of the specific detection of Xanthomonas phaseoli pv. manihotis based on the pthB gene

2019 ◽  
Vol 41 ◽  
pp. e42708
Author(s):  
Rita de Cássia Cerqueira Melo ◽  
Carlos Augusto Dorea Bragança ◽  
Katia Nogueira Pestana ◽  
Harllen Sandro Alves da Silva ◽  
Claudia Fortes Ferreira ◽  
...  
Keyword(s):  
Universal Primers ◽  
Rrna Gene ◽  
Reverse Primer ◽  
Validation Procedure ◽  
Proposed Modification ◽  
Different Strains ◽  
Pcr Conditions ◽  
Forward Primer ◽  
Made In

Modifications were made in the PCR conditions aiming to overcome the problem of non-amplification of the Xanthomonas phaseoli pv. manihotis (Xpm) fragment, using the primer pair XV / XK described in the literature. The objective of this study was to propose changes in the primers already described (XV / XK_MOD) and validate the use of these new primers in identifying Xpm. The validation procedure was carried out with the primer pair XV and XK_MOD, using different strains of Xpm, other plant pathogenic and endophytic bacteria genera and one isolate of X. phaseoli pv. passiflorae. As a control, additional reactions were conducted in multiplex with the universal primers for the 16S rRNA gene of the bacteria together with XV / XK and XV / XK_MOD. Using the forward primer (XV) described in the literature together with the modified reverse primer (XK_MOD), it was possible to achieve amplification from DNA extracted from in vitro cultures and from infected tissue, but no amplification was noticed for the primer pair described in the literature, confirming the effectiveness of the proposed modification.

scholarly journals Novel Clade of Rickettsia spp. from Leeches

2002 ◽  
Vol 68 (2) ◽  
pp. 999-1004 ◽  
Author(s):  
Yoshitomo Kikuchi ◽  
Shinya Sameshima ◽  
Osamu Kitade ◽  
Junichi Kojima ◽  
Takema Fukatsu
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Segment ◽  
Natural Populations ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Specific Primers ◽  
Diagnostic Pcr ◽  
Transmission Electron ◽  
The 16S Rrna Gene

ABSTRACT Intracellular rickettsia-like structures were found in the tissues of a glossiphoniid leech, Torix tagoi, by transmission electron microscopy. Diagnostic PCR analysis using specific primers suggested that of the nine glossiphoniid species examined, two species, T. tagoi and Hemicrepsis marginata, harbored bacteria of the genus Rickettsia. A 1.5-kb eubacterial 16S rRNA gene segment obtained from each of these species was amplified by PCR, cloned, and sequenced. Phylogenetic analysis of the 16S rRNA gene demonstrated that the Rickettsia species found in the leeches constituted a novel clade that is distinct from the clade of arthropod-associated Rickettsia species. In natural populations, 97.7% (43 of 44) of T. tagoi leeches and 100% (9 of 9) of H. marginata leeches carried Rickettsia, suggesting that infection with Rickettsia is prevalent in these leeches. This is the first report of Rickettsia found in annelids.

scholarly journals A Differential Metabarcoding Approach to Describe Taxonomy Profiles of Bacteria and Archaea in the Saltern of Margherita di Savoia (Italy)

2020 ◽  
Vol 8 (6) ◽  
pp. 936 ◽  
Author(s):  
Claudia Leoni ◽  
Mariateresa Volpicella ◽  
Bruno Fosso ◽  
Caterina Manzari ◽  
Elisabetta Piancone ◽  
...  
Keyword(s):  
Ecological Model ◽  
Salinity Range ◽  
Rrna Gene ◽  
Hypervariable Regions ◽  
Cell Counts ◽  
Precious Resource ◽  
Catalyzed Reporter Deposition ◽  
Card Fish ◽  
The 16S Rrna Gene

Microorganisms inhabiting saline environments are an interesting ecological model for the study of the adaptation of organisms to extreme living conditions and constitute a precious resource of enzymes and bioproducts for biotechnological applications. We analyzed the microbial communities in nine ponds with increasing salt concentrations (salinity range 4.9–36.0%) of the Saltern of Margherita di Savoia (Italy), the largest thalassohaline saltern in Europe. A deep-metabarcoding NGS procedure addressing separately the V5-V6 and V3-V4 hypervariable regions of the 16S rRNA gene of Bacteria and Archaea, respectively, and a CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) analysis allowed us to profile the dynamics of microbial populations at the different salt concentrations. Both the domains were detected throughout the saltern, even if the low relative abundance of Archaea in the three ponds with the lowest salinities prevented the construction of the relative amplicon libraries. The highest cell counts were recorded at 14.5% salinity for Bacteria and at 24.1% salinity for Archaea. While Bacteria showed the greatest number of genera in the first ponds (salinity range 4.9–14.5%), archaeal genera were more numerous in the last ponds of the saltern (salinity 24.1–36.0%). Among prokaryotes, Salinibacter was the genus with the maximum abundance (~49% at 34.6% salinity). Other genera detected at high abundance were the archaeal Haloquadratum (~43% at 36.0% salinity) and Natronomonas (~18% at 13.1% salinity) and the bacterial “Candidatus Aquiluna” (~19% at 14.5% salinity). Interestingly, “Candidatus Aquiluna” had not been identified before in thalassohaline waters.

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