scholarly journals Use of Multiplex Terminal Restriction Fragment Length Polymorphism for Rapid and Simultaneous Analysis of Different Components of the Soil Microbial Community▿

2006 ◽  
Vol 72 (11) ◽  
pp. 7278-7285 ◽  
Author(s):  
Brajesh K. Singh ◽  
Loic Nazaries ◽  
Stacey Munro ◽  
Ian C. Anderson ◽  
Colin D. Campbell
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Microbial Communities ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Soil Samples ◽  
Pine Forest ◽  
Pcr Products ◽  
Restriction Fragment Length

ABSTRACT A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.

T-BAPS: A Bayesian Statistical Tool for Comparison of Microbial Communities Using Terminal-restriction Fragment Length Polymorphism (T-RFLP) Data

2007 ◽  
Vol 6 (1) ◽  
Author(s):  
Jing Tang ◽  
Jinglun Tao ◽  
Hidetoshi Urakawa ◽  
Jukka Corander
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Microbial Communities ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Bayesian Model ◽  
Missing Values ◽  
Latent Class ◽  
Fragment Length Polymorphism ◽  
Restriction Fragment Length

The investigation of microbial communities is an essential part of the study of the biosphere. Flexible molecular fingerprinting tools such as terminal-restriction fragment length polymorphism (T-RFLP) analysis are often applied in the studies to enable the characterization of the microbial population. However, such data have so far been primarily analyzed using conventional clustering methods. Here we introduce a Bayesian model-based method for the purpose of comparing microbial communities using T-RFLP data. Such datasets have in general several challenging features, e.g. sparseness, missing values and structurally zero-valued observations. These features are taken into account by developing a Bayesian latent class mixture model for the observations in our framework. To make inferences under the model we use a recent Markov chain Monte Carlo (MCMC) -based method for the Bayesian model selection. To assess the introduced method we analyze both simulated and real datasets. The simulations show that our approach compares preferably to standard statistical clustering tools, such as k-means, hierarchical clustering, and Autoclass. The developed tool is freely available as a software package T-BAPS at http://www.abo.fi/fak/mnf/mate/jc/software/t-baps.html.

scholarly journals Rapid Identification and Typing ofStaphylococcus aureus by PCR-Restriction Fragment Length Polymorphism Analysis of the aroA Gene

1999 ◽  
Vol 37 (3) ◽  
pp. 570-574 ◽  
Author(s):  
Javier Yugueros Marcos ◽  
Alberto Cascón Soriano ◽  
María Sánchez Salazar ◽  
Carmen Hernanz Moral ◽  
Susana Suárez Ramos ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rapid Identification ◽  
Polymorphism Analysis ◽  
Pcr Products ◽  
Dna Fragment ◽  
Restriction Fragment Length

The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when other Staphylococcus spp. or genera were analyzed by using the same pair of primers. The detection limit for S. aureus cells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 × 102 CFU) when S. aureus cells were suspended in sterilized whole milk. TaqI digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.

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