DNA Heteropolymorphism of Chum Salmon Detected by Denaturing Gradient Gel Electrophoresis and Real Time PCR

ABSTRACT The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.

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Expression of thenifHgene in diazotrophic bacteria inEucalyptus urograndisplantations

Canadian Journal of Forest Research ◽

10.1139/cjfr-2015-0063 ◽

2016 ◽

Vol 46 (2) ◽

pp. 190-199 ◽

Cited By ~ 3

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.

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Degradation of 4-Chloro-2-Methylphenoxyacetic Acid in Top- and Subsoil Is Quantitatively Linked to the Class III tfdA Gene

Applied and Environmental Microbiology ◽

10.1128/aem.72.2.1476-1486.2006 ◽

2006 ◽

Vol 72 (2) ◽

pp. 1476-1486 ◽

Cited By ~ 68

Author(s):

Jacob Bælum ◽

Trine Henriksen ◽

Hans Christian Bruun Hansen ◽

Carsten Suhr Jacobsen

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Denaturing Gradient Gel Electrophoresis ◽

Pcr Amplification ◽

Dry Weight ◽

Initial Assessment ◽

Class Iii ◽

Gradient Gel Electrophoresis ◽

Pcr Method ◽

Or Genes

ABSTRACT The tfdA gene is known to be involved in the first step of the degradation of the phenoxy acid herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in several soil bacteria, but bacteria containing other tfdA-like genes have been isolated as well. A quantitative real-time PCR method was used to monitor the increase in the concentration of tfdA genes during degradation of MCPA in sandy topsoil and subsoil over a period of 115 days. Quantitative PCR revealed growth in the tfdA-containing bacterial community, from 500 genes g−1 soil to approximately 3 × 104 genes g−1 soil and to 7 × 105 genes g−1 soil for topsoil initially added to 2.3 mg MCPA kg−1 (dry weight) soil and 20 mg MCPA kg−1 (dry weight) soil, respectively. We analyzed the diversity of the tfdA gene during the degradation experiment. Analyses of melting curves of real-time PCR amplification products showed that a shift in the dominant tfdA population structure occurred during the degradation period. Further denaturing gradient gel electrophoresis and sequence analysis revealed that the tfdA genes responsible for the degradation of MCPA belonged to the class III tfdA genes, while the tfdA genes present in the soil before the occurrence of degradation belonged to the class I tfdA genes. The implications of these results is that the initial assessment of functional genes in soils does not necessarily reflect the organisms or genes that would carry out the degradation of the compounds in question.

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Microbiota during fermentation of chum salmon (Oncorhynchus keta) sauce mash inoculated with halotolerant microbial starters: Analyses using the plate count method and PCR-denaturing gradient gel electrophoresis (DGGE)

Food Microbiology ◽

10.1016/j.fm.2009.12.008 ◽

2010 ◽

Vol 27 (4) ◽

pp. 509-514 ◽

Cited By ~ 20

Author(s):

Shuji Yoshikawa ◽

Daisuke Yasokawa ◽

Koji Nagashima ◽

Koji Yamazaki ◽

Hideyuki Kurihara ◽

...

Keyword(s):

Gel Electrophoresis ◽

Chum Salmon ◽

Denaturing Gradient Gel Electrophoresis ◽

Plate Count ◽

Oncorhynchus Keta ◽

Chum Salmon Oncorhynchus Keta ◽

Count Method ◽

Gradient Gel Electrophoresis ◽

Plate Count Method

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Bacterial population dynamics and community structure in a pharmaceutical manufacturing water supply system determined by real-time PCR and PCR-denaturing gradient gel electrophoresis

Journal of Applied Microbiology ◽

10.1111/j.1365-2672.2004.02396.x ◽

2004 ◽

Vol 97 (6) ◽

pp. 1123-1131 ◽

Cited By ~ 13

Author(s):

M. Kawai ◽

J. Yamagishi ◽

N. Yamaguchi ◽

K. Tani ◽

M. Nasu

Keyword(s):

Population Dynamics ◽

Community Structure ◽

Water Supply ◽

Real Time Pcr ◽

Gel Electrophoresis ◽

Bacterial Population ◽

Denaturing Gradient Gel Electrophoresis ◽

Supply System ◽

Pharmaceutical Manufacturing ◽

Gradient Gel Electrophoresis

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Molecular diagnostics of periodontitis

Postępy Higieny i Medycyny Doświadczalnej ◽

10.5604/01.3001.0010.3789 ◽

2017 ◽

Vol 71 (1) ◽

pp. 47-56

Author(s):

Izabela Korona-Głowniak ◽

Radosław Siwiec ◽

Marcin Berger ◽

Anna Malm ◽

Jolanta Szymańska

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Molecular Diagnostics ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Chain Reaction ◽

Gradient Gel Electrophoresis ◽

Polymerase Chain ◽

Temperature Gradient Gel Electrophoresis ◽

Complex Relationships

The microorganisms that form dental plaque are the main cause of periodontitis. Their identification and the understanding of the complex relationships and interactions that involve these microorganisms, environmental factors and the host’s health status enable improvement in diagnostics and targeted therapy in patients with periodontitis. To this end, molecular diagnostics techniques (both techniques based on the polymerase chain reaction and those involving nucleic acid analysis via hybridization) come increasingly into use. On the basis of a literature review, the following methods are presented: polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time PCR), 16S rRNA-encoding gene sequencing, checkerboard and reverse-capture checkerboard hybridization, microarrays, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), as well as terminal restriction fragment length polymorphism (TRFLP) and next generation sequencing (NGS). The advantages and drawbacks of each method in the examination of periopathogens are indicated. The techniques listed above allow fast detection of even small quantities of pathogen present in diagnostic material and prove particularly useful to detect microorganisms that are difficult or impossible to grow in a laboratory.

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