Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil VSports app下载. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

Comparative Study
. 2004 Oct;70(10):6147-56.
doi: 10.1128/AEM.70.10.6147-6156.2004.

Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities

Massimiliano Cardinale  1 , Lorenzo Brusetti, Paola Quatrini, Sara Borin, Anna Maria Puglia, Aurora Rizzi, Elisabetta Zanardini, Claudia Sorlini, Cesare Corselli, Daniele Daffonchio
Affiliations
Comparative Study

Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities

"V体育官网入口" Massimiliano Cardinale et al. Appl Environ Microbiol. 2004 Oct.

Abstract

ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M. M. Fisher and E. W. Triplett, Appl. Environ. Microbiol. 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L. Ranjard et al. , Appl. Environ. Microbiol. 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the facade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets. The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets. The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments. The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species. ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0. 14 ng microl(-1), while the other primer sets failed to detect the spacers of one or more bacterial strains. Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri. VSports手机版.

PubMed Disclaimer

VSports - Figures

FIG. 1.
FIG. 1.
Positions of the six primers on the prokaryote ribosomal operon and numbers of nucleotides belonging to 16S and 23S rRNA genes amplified by PCR. The numbers of nucleotides amplified on 16S and 23S rRNA genes and the positions of the annealing sites by each primer set were calculated based on the E. coli O157:H7 ribosomal genes. (A) ITSF/ITSReub; (B) SD-Bact-1522-b-S-20/LD-Bact-132-a-A-18 (19); (C) 1406F/23Sr (4).
FIG. 2.
FIG. 2.
Comparison of the relative areas of the peaks produced by the three primer sets in the ARISA of a model community containing 280, 28, 2.8, 1.4, or 0.14 ng of DNA of six bacterial reference strains. Black bar, B. cereus 31T; dark grey bar, S. coelicolor M145; light grey bar, P. stutzeri 1015; dotted bar, S. bovis JM300; lined bar, E. coli DSM 50902; white bar, A. undicula USDA 4903. The expected peak areas, corresponding to the DNA fractions in the model community, should range between 6.0 and 45.2% of the total fluorescence.
FIG. 3.
FIG. 3.
Relative areas of the peaks corresponding to the spacer of P. stutzeri 1015 and A. undicula USDA 4903 produced by the three primer sets when the strain DNA represented fractions of 50% (vertically lined bar), 9.1% (horizontally lined bar), 4.8% (dotted bar), 1.0% (grey bar), 0.5% (white bar), and 0.1% (black bar) of a model community DNA including three other bacterial species (B. cereus, E. coli, and S. coelicolor).
See this image and copyright information in PMC

References

    1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410. - PubMed (V体育2025版)
    1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1994. Current protocols of molecular biology. John Wiley & Sons, Inc., New York, N.Y.
    1. Blackwood, C. B., T. Marsh, S.-H. Kim, and E. A. Paul. 2003. Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities. Appl. Environ. Microbiol. 69:926-932. - PMC - PubMed
    1. Borneman, J., and E. W. Triplett. 1997. Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and population shifts associated with deforestation. Appl. Environ. Microbiol. 63:2647-2653. - PMC - PubMed
    1. Cherif, A., S. Borin, A. Rizzi, H. Ouzari, A. Boudabous, and D. Daffonchio. 2003. Bacillus anthracis diverges from related clades of the Bacillus cereus group in 16S-23S ribosomal DNA intergenic transcribed spacers containing tRNA genes. Appl. Environ. Microbiol. 69:33-40. - PMC - PubMed

MeSH terms (V体育官网)

LinkOut - more resources