scholarly journals Effects of orally administeredLactobacillus caseiDN-114 001 on the composition or activities of the dominant faecal microbiota in healthy humans

2006 ◽  
Vol 95 (2) ◽  
pp. 421-429 ◽  
Author(s):  
Violaine Rochet ◽  
Lionel Rigottier-Gois ◽  
Maléne Sutren ◽  
Marie-Noëlle Krementscki ◽  
Claude Andrieux ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fold Increase ◽  
Fermented Milk ◽  
Milk Product ◽  
Faecal Microbiota ◽  
Specific Primers ◽  
Healthy Humans ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis

The composition and activities of the faecal microbiota in twelve healthy subjects analysed in a single open study were monitored before (1-week baseline step), during (10d supplementation step) and after (10d follow-up step) the ingestion of a fermented milk containingLactobacillus caseiDN-114001. Fluorescentin situhybridisation with group-specific DNA probes, real-time PCR usingL. paracaseigroup-specific primers and temporal temperature gradient gel electrophoresis (TTGE) using group-specific primers were carried out, together with bacterial enzyme activity and metabolite analyses to monitor the structure and activities of the faecal microbiota.L. caseiDNA was detected in the faeces of all of the subjects by TTGE after 10d supplementation. Its quantification by real-time PCR showed a 1000-fold increase during the test step compared with initial levels. No major modification in either the dominant members of the faecal microbiota or their activities was observed during the trial. In conclusion, the short-term consumption of a milk product containingL. caseiDN-114001 was accompanied by a high, transient increase in the quantity of this strain in the faeces of all of the subjects without markedly affecting biochemical or bacteriological factors.

scholarly journals Influence of Camembert consumption on the composition and metabolism of intestinal microbiota: a study in human microbiota-associated rats

2004 ◽  
Vol 92 (3) ◽  
pp. 429-438 ◽  
Author(s):  
Christophe Lay ◽  
Malène Sutren ◽  
Pascale Lepercq ◽  
Catherine Juste ◽  
Lionel Rigottier-Gois ◽  
...  
Keyword(s):  
Intestinal Microbiota ◽  
Dairy Products ◽  
Basal Diet ◽  
Fermented Milk ◽  
Metabolic Characteristics ◽  
Human Microbiota ◽  
Gradient Gel Electrophoresis ◽  
Metabolic Activities ◽  
Temperature Gradient Gel Electrophoresis ◽  
Micro Organisms

The objective of the present study was to evaluate the consequence of Camembert consumption on the composition and metabolism of human intestinal microbiota. Camembert cheese was compared with milk fermented by yoghurt starters andLactobacillus caseias a probiotic reference. The experimental model was the human microbiota-associated (HM) rat. HM rats were fed a basal diet (HMB group), a diet containing Camembert made from pasteurised milk (HMCp group) or a diet containing fermented milk (HMfm group). The level of micro-organisms from dairy products was measured in faeces using cultures on a specific medium and PCR–temporal temperature gradient gel electrophoresis. The metabolic characteristics of the caecal microbiota were also studied: SCFA, NH3, glycosidase and reductase activities, and bile acid degradations. The results showed that micro-organisms from cheese comprised 105–108bacteria/g faecal sample in the HMCp group.Lactobacillusspecies from fermented milk were detected in HMfm rats. Consumption of cheese and fermented milk led to similar changes in bacterial metabolism: a decrease in azoreductase activity and NH3concentration and an increase in mucolytic activities. However, specific changes were observed: in HMCp rats, the proportion of ursodeoxycholic resulting from chenodeoxycholic epimerisation was higher; in HMfm rats, α and β-galactosidases were higher than in other groups and both azoreductases and nitrate reductases were lower. The results show that, as for fermented milk, Camembert consumption did not greatly modify the microbiota profile or its major metabolic activities. Ingested micro-organisms were able to survive in part during intestinal transit. These dairy products exert a potentially beneficial influence on intestinal metabolism.

scholarly journals Expression of thenifHgene in diazotrophic bacteria inEucalyptus urograndisplantations

2016 ◽  
Vol 46 (2) ◽  
pp. 190-199 ◽  
Author(s):  
Marliane de Cássia Soares da Silva ◽  
Igor Rodrigues Mendes ◽  
Thiago de Almeida Paula ◽  
Roberto Sousa Dias ◽  
Sérgio Oliveira de Paula ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Nifh Gene ◽  
Root Systems ◽  
N Fertilization ◽  
Quantitative Real Time Pcr ◽  
Diazotrophic Bacteria ◽  
Eucalypt Plantations ◽  
Gradient Gel Electrophoresis

A large proportion of eucalypt plantations in Brazil are located in areas with low soil fertility. The actions of microorganisms are of great importance for the cycling of nutrients, including nitrogen (N), that are essential for plant metabolism. Denaturing gradient gel electrophoresis (DGGE) was used to monitor and identify the total and active microorganisms involved in the N cycle in both the soil and root systems of a forest of Eucalyptus urograndis with sections that were fertilized with N or unfertilized. Quantitative real-time PCR was used to examine the expression of the nifH gene in N-fixing bacteria present in both the soil and root systems. According to the DGGE analysis, in the total and active populations of N-fixing bacteria, the presence and expression of the nifH gene were influenced by the winter and summer seasons and (or) N fertilization, respectively. DGGE band sequencing from total DNA samples showed that the most abundant group of diazotrophic bacteria belonged to Alphaproteobacteria in both the soil and root systems. Quantitative real-time PCR revealed that nifH expression was higher in the soil samples, especially in those that did not receive N fertilization. The differences in the composition of the total and active diazotrophic populations highlight the importance of evaluating the active populations, because they are effectively responsible for the biogeochemical transformation of N and also control its’ availability to plants.

scholarly journals Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

scholarly journals Co-infection of bovine papillomavirus type-1 and -10 in teat warts of dairy cattle

2013 ◽  
Vol 58 (No. 12) ◽  
pp. 605-608
Author(s):  
P. Kumar ◽  
BL Jangir ◽  
G. Saikumar ◽  
R. Somvanshi
Keyword(s):  
Dairy Cattle ◽  
Real Time ◽  
Real Time Pcr ◽  
Rice Grain ◽  
Bovine Papillomavirus ◽  
Viral Dna ◽  
Specific Primers ◽  
Copy Numbers ◽  
Pcr Techniques

The present study was carried out to investigate the involvement of different bovine papillomaviruses in the teat warts of cattle. A total of 11 teat wart samples showing rice grain-like and small, sessile elevated greyish or flesh-like growths were collected from dairy cattle. DNA was extracted from these teat wart samples and PCR and real time PCR techniques were applied using specific primers for BPV-1 and -10 to detect the presence of viral nucleic acid. PCR revealed the presence of viral DNA of BPV-1 and -10 in three and seven samples, respectively. Quantification using real time PCR revealed that the copy numbers of the viral DNA of BPV-1 and -10 DNA varied from 1.12E + 04 to 2.99E + 04 and 3.56E + 02 to 5.23E + 06, respectively. From the present study it can be concluded that BPV-1 and -10 are involved in production of rice grain-like and sessile elevated growths on the teats of cattle.

Real-time PCR Assay with SNP-specific Primers for the Detection of a G143A Mutation Level in Venturia inaequalis Field Populations

2011 ◽  
Vol 159 (7-8) ◽  
pp. 569-578 ◽  
Author(s):  
Monika Michalecka ◽  
Tadeusz Malinowski ◽  
Agata Broniarek-Niemiec ◽  
Anna Bielenin
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Venturia Inaequalis ◽  
Pcr Assay ◽  
Specific Primers

scholarly journals Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

2004 ◽  
Vol 70 (1) ◽  
pp. 167-173 ◽  
Author(s):  
Takahiro Matsuki ◽  
Koichi Watanabe ◽  
Junji Fujimoto ◽  
Yukiko Kado ◽  
Toshihiko Takada ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Intestinal Flora ◽  
Healthy Adults ◽  
Pcr Detection ◽  
Distribution Analysis ◽  
Specific Primers ◽  
Cell Counts ◽  
Species Specific

ABSTRACT A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 106 to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >106 cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.

Differentiation of infectious bronchitis virus vaccine strains Ma5 and 4/91 by TaqMan real-time PCR

2017 ◽  
Vol 20 (3) ◽  
pp. 599-601 ◽  
Author(s):  
T. Stenzel ◽  
D. Dziewulska ◽  
M. Śmiałek ◽  
B. Tykałowski ◽  
J. Kowalczyk ◽  
...  
Keyword(s):  
Real Time ◽  
Virus Vaccine ◽  
Infectious Bronchitis Virus ◽  
Real Time Pcr ◽  
Cross Reactivity ◽  
Specific Primers ◽  
Assay Sensitivity ◽  
Infectious Bronchitis ◽  
Molecular Assays ◽  
Rapid Molecular Assays

Abstract The aim of this study was to develop rapid molecular assays for differentiating vaccine strains Ma5 and 4/91 of the infectious bronchitis virus (IBV). Specific primers and probes for S1 and N genes were designed based on the nucleotide sequences of both vaccine strains. Cross-reactivity was not observed. Assay sensitivity was 2.373 × 103 copies of the Ma5 strain, and 3.852 x 103 copies of the 4/91 strain. Samples belonging to a known genotype demonstrated that the designed assays supported rapid and sensitive detection of Ma5 and 4/91 vaccine strains of IBV.

scholarly journals Comparison of Reverse Transcription Quantitative Real-Time PCR, Flow Cytometry, and Immunohistochemistry for Detection of Monoclonality in Lymphomas

ISRN Oncology ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-6
Author(s):  
Anders Ståhlberg ◽  
Pierre Åman ◽  
Linda Strömbom ◽  
Neven Zoric ◽  
Alfredo Diez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Real Time ◽  
B Lymphocytes ◽  
Light Chain ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Light Chains ◽  
Quantitative Real Time Pcr ◽  
Healthy Humans

In healthy humans, 60–70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa : lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas. Tumor biopsies from 55 patients with B cell malignancies, 5 B-lymphoid tumor cell lines, and 20 biopsies from patients with lymphadenitis were analyzed by immunohistochemistry, flow cytometry, and reverse transcription quantitative real-time PCR. Clonality was determined by immunohistochemistry in 52/53 cases, flow cytometry in 30/39 cases, and reverse transcription quantitative real-time PCR in 33/55 cases. In conclusion, immunohistochemistry was superior to flow cytometry and reverse transcription quantitative real-time PCR for clonality identification. Flow cytometry and reverse transcription quantitative real-time PCR analysis has complementary values. In a considerable number of cases tumor cells produced both kappa and lambda light chain transcripts, but only one type of light chain peptide was produced.

Evaluation of temperature gradient gel electrophoresis for the analysis of prey DNA within the guts of invertebrate predators

2006 ◽  
Vol 96 (3) ◽  
pp. 295-304 ◽  
Author(s):  
G.L. Harper ◽  
S.K. Sheppard ◽  
J.D. Harwood ◽  
D.S. Read ◽  
D.M. Glen ◽  
...  
Keyword(s):  
Temperature Gradient ◽  
Gel Electrophoresis ◽  
Gut Contents ◽  
Generalist Predators ◽  
Specific Primers ◽  
Banding Patterns ◽  
Field Samples ◽  
Gradient Gel Electrophoresis ◽  
Pterostichus Melanarius ◽  
Temperature Gradient Gel Electrophoresis

AbstractThe utility of temperature gradient gel electrophoresis (TGGE) as a means of analysing the gut contents of predators was evaluated. Generalist predators consume multiple prey species and a species-specific primer approach may not always be a practical means of analysing predator responses to prey diversity in complex and biodiverse ecosystems. General invertebrate primers were used to amplify the gut contents of predators, generating banding patterns that identified component prey remains. There was no evidence of dominance of the polymerase chain reaction (PCR) by predator DNA. When applied to field samples of the carabid predatorPterostichus melanarius(Illiger) nine banding patterns were detected, including one for aphids. To further distinguish between species, group-specific primers were designed to separate species of earthworm and aphid. TGGE of the earthworm PCR products generated banding patterns that varied with haplotype in some species. Aphid and earthworm DNA could be detected in the guts of carabids for up to 24 h using TGGE. InP. melanarius, with low numbers of prey per insect gut (mean < 3), interpretation of banding patterns proved to be tractable. Potential problems of interpretation of TGGE gels caused by multiple prey bands, cryptic bands, haplotype variation, taxonomic uncertainties (especially with regard to earthworms), secondary predation, scavenging and presence of parasites and parasitoids in the prey or the predators, are discussed. The results suggest that PCR, using combinations of general invertebrate and group-specific primers followed by TGGE, provides a potentially useful approach to the analysis of multiple uncharacterized prey in predators.

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