16S rRNA gene-based analysis of microbial community by whole-genome amplification and minigel-single-strand conformation polymorphism technique

Oto, Michiei; Suda, Wataru; Shinoyama, Hirofumi

doi:10.1263/jbb.102.482

Cited by 6 publications

(3 citation statements)

References 15 publications

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“…Whole genome amplification (WGA) using phi29 DNA polymerase overcomes insufficient sample sizes [ 12 ]. For example, numerous studies of microbial ecology show that WGA using phi29 DNA polymerase enriches environmental template DNAs with the highest amplification efficiency [ 13 , 14 ], and the amplified DNA can be used to characterize the microbial community structure in low-biomass environments [ 15 – 17 ]. Here, we applied phi29 DNA polymerase to generate sufficient quantities of DNA from oropharyngeal mucosal swabs to analyze and monitor the microbial community associated with liver cirrhosis and pneumonia, which was accompanied by further DGGE analysis of the V3 region of the 16S rDNA.…”

Section: Introductionmentioning

confidence: 99%

Alterations of Bacteroides sp., Neisseria sp., Actinomyces sp., and Streptococcus sp. populations in the oropharyngeal microbiome are associated with liver cirrhosis and pneumonia

Lü

Qian²,

Ren

et al. 2015

BMC Infect Dis

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BackgroundThe microbiomes of humans are associated with liver and lung inflammation. We identified and verified alterations of the oropharyngeal microbiome and assessed their association with cirrhosis and pneumonia.MethodsStudy components were as follows: (1) determination of the temporal stability of the oropharyngeal microbiome; (2) identification of oropharyngeal microbial variation in 90 subjects; (3) quantitative identification of disease-associated bacteria. DNAs enriched in bacterial sequences were produced from low-biomass oropharyngeal swabs using whole genome amplification and were analyzed using denaturing gradient gel electrophoresis analysis.ResultsWhole genome amplification combined with denaturing gradient gel electrophoresis analysis monitored successfully oropharyngeal microbial variations and showed that the composition of each subject’s oropharyngeal microbiome remained relatively stable during the follow-up. The microbial composition of cirrhotic patients with pneumonia differed from those of others and clustered together in subgroup analysis. Further, species richness and the value of Shannon’s diversity and evenness index increased significantly in patients with cirrhosis and pneumonia versus others (p < 0.001, versus healthy controls; p < 0.01, versus cirrhotic patients without pneumonia). Moreover, we identified variants of Bacteroides, Eubacterium, Lachnospiraceae, Neisseria, Actinomyces, and Streptococcus through phylogenetic analysis. Quantitative polymerase chain reaction assays revealed that the populations of Bacteroides, Neisseria, and Actinomycetes increased, while that of Streptococcus decreased in cirrhotic patients with pneumonia versus others (p < 0.001, versus Healthy controls; p < 0.01, versus cirrhotic patients without pneumonia).ConclusionsAlterations of Bacteroides, Neisseria, Actinomyces, and Streptococcus populations in the oropharyngeal microbiome were associated with liver cirrhosis and pneumonia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-0977-x) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Alterations of Bacteroides sp., Neisseria sp., Actinomyces sp., and Streptococcus sp. populations in the oropharyngeal microbiome are associated with liver cirrhosis and pneumonia

Lü

Qian²,

Ren

et al. 2015

BMC Infect Dis

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“…Amplification using DNA extracted from a low amount of cells (0.05 μl blood) can eliminate interference due to the presence of inhibitory factors [ 46 ]. It has been supported by other studies which involve human clinical specimens [ 13 ] and freshly cultured bacteria [ 18 , 20 – 22 , 31 , 32 , 47 ]. The selection of method for DNA extraction for PCMs can be determined based on the amount or sample volume.…”

Section: Discussionmentioning

confidence: 54%

A rapid and sensitive system for recovery of nucleic acids from Mycobacteria sp. on archived glass slides

Talip

Snelling²,

Sleator

et al. 2018

BMC Microbiol

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BackgroundThe field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM coupled with PCR amplification of a suitable target.ResultsMycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM.ConclusionsThis study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system.

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“…In recent years, molecular biological techniques have been used to study environmental microbial community structures. Such techniques include PCR-denaturing gradient gel electrophoresis (DGGE) and the PCR-single strand conformation polymorphism (SSCP) method 11,19,28) . For these techniques, PCR inhibitor-free DNA should be prepared by simple, rapid, and cost-effective methods.…”

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confidence: 99%

A Direct Method to Isolate DNA from Phyllosphere Microbial Communities without Disrupting Leaf Tissues

et al. 2008

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We developed a method for direct DNA isolation from phyllosphere microbial communities, designated Direct-DIP. This method comprises DNA extraction from non-shredded leaves with benzyl chloride, and DNA purification by gel filtration. Scanning electron microscopy showed that epiphytic microorganisms were completely removed from the leaf surface after benzyl chloride treatment, while microstructures of the leaf were not damaged. Clear DGGE profiles were obtained regardless of the plant species. Shannon diversity indices of DGGE profiles by Direct-DIP were higher than those by a conventional method. Our findings suggest that Direct-DIP is a rapid, simple, and cost-effective method of extracting DNA from phyllosphere microbial communities.

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16S rRNA gene-based analysis of microbial community by whole-genome amplification and minigel-single-strand conformation polymorphism technique

Cited by 6 publications

References 15 publications

Alterations of Bacteroides sp., Neisseria sp., Actinomyces sp., and Streptococcus sp. populations in the oropharyngeal microbiome are associated with liver cirrhosis and pneumonia

Alterations of Bacteroides sp., Neisseria sp., Actinomyces sp., and Streptococcus sp. populations in the oropharyngeal microbiome are associated with liver cirrhosis and pneumonia

A rapid and sensitive system for recovery of nucleic acids from Mycobacteria sp. on archived glass slides

A Direct Method to Isolate DNA from Phyllosphere Microbial Communities without Disrupting Leaf Tissues

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