Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities
Articolo
Data di Pubblicazione:
2004
Citazione:
Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities / M. Cardinale, L. Brusetti, P.Quatrini, S. Borin, A.M. Puglia, A. Rizzi, E. Zanardini, C. Sorlini, C. Corselli, D. Daffonchio. - In: APPLIED AND ENVIRONMENTAL MICROBIOLOGY. - ISSN 0099-2240. - 70:10(2004), pp. 6147-6156.
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 fac¸ade 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 l 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.
Tipologia IRIS:
01 - Articolo su periodico
Elenco autori:
M. Cardinale, L. Brusetti, P.Quatrini, S. Borin, A.M. Puglia, A. Rizzi, E. Zanardini, C. Sorlini, C. Corselli, D. Daffonchio
Link alla scheda completa: