| 1. |
- Sipos, Rita, et al.
(författare)
-
Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis
- 2007
-
Ingår i: FEMS Microbiology Ecology. - : Oxford University Press (OUP). - 0168-6496 .- 1574-6941. ; 60:2, s. 341-350
-
Tidskriftsartikel (refereegranskat)abstract
- In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using ’universal’ bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61°C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR. © 2007 Federation of European Microbiological Societies.
|
|
| 2. |
- Székely, Anna J., et al.
(författare)
-
DGGE and T-RFLP analysis of bacterial succession during mushroom compost production and sequence-aided T-RFLP profile of mature compost
- 2009
-
Ingår i: Microbial Ecology. - : Springer Science and Business Media LLC. - 0095-3628 .- 1432-184X. ; 57:3, s. 522-533
-
Tidskriftsartikel (refereegranskat)abstract
- The amount of button mushroom (Agaricus bisporus) harvested from compost is largely affected by the microbial processes taking place during composting and the microbes inhabiting the mature compost. In this study, the microbial changes during the stages of this specific composting process were monitored, and the dominant bacteria of the mature compost were identified to reveal the microbiological background of the favorable properties of the heat-treated phase II mushroom compost. 16S ribosomal deoxyribonucleic acid (rDNA)-based denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) molecular fingerprinting methods were used to track the succession of microbial communities in summer and winter composting cycles. DNA from individual DGGE bands were reamplified and subjected to sequence analysis. Principal component analysis of fingerprints of the composting processes showed intensive changes in bacterial community during the 22-day procedure. Peak temperature samples grouped together and were dominated by Thermus thermophilus. Mature compost patterns were almost identical by both methods (DGGE, T-RFLP). To get an in-depth analysis of the mature compost bacterial community, the sequence data from cultivation of the bacteria and cloning of environmental 16S rDNA were uniquely coupled with the output of the environmental T-RFLP fingerprints (sequence-aided T-RFLP). This method revealed the dominance of a supposedly cellulose-degrading consortium composed of phylotypes related to Pseudoxanthomonas, Thermobifida, and Thermomonospora. © 2008 Springer Science+Business Media, LLC.
|
|
