Ribosomal intergenic spacer analysis (RISA) was used to analyze the structure of microbial populations in soil (4
) by comparing the profiles after polyacrylamide gel electrophoresis. At most, only a few tens of bands were detected, suggesting a probable underestimation of diversity due to difficulty in identifying weak bands and resolving contiguous bands. The automated version of RISA, the ARISA, is a rapid and precise technique that allows microbial communities to be investigated and compared easily, highlighting the taxonomic diversity, evident from the marked variability in ribosomal spacer length, in the prokaryote genomes (7
). ARISA explores microbial diversity at the intraspecific level (5
) and can be considered one of the most suitable techniques for rapidly analyzing and comparing great numbers of samples. Considering the very interesting potential of large-scale ARISA applications, we deemed it suitable for carrying out a standardization of the technique in order to permit an easy comparison of results from different studies.
An important step in standardizing PCR-based methods for the analysis of bacterial communities is to choose a primer set that can strongly achieve reliability in the results. We have compared the performance of two primer sets published for performing ARISA with a newly designed primer set. This new ITSF/ITSReub primer set yielded higher peaks than primer sets S-D-Bact-1522-b-S-20/L-D-Bact-132-a-A-18 and 1406F and 23Sr. Moreover, the primer set 1406F/23Sr was not able to amplify the internal transcribed spacers (ITS) of R. leguminosarum USDA 2370 and of S. meliloti 1002. Search for homology of the two primers for the 16S and 23S rRNA gene sequence of R. leguminosarum revealed that the forward primer has mismatches at the 3′ ends with 16S rRNA genes of R. leguminosarum. This defective annealing, coupled to the length of the ITS of these strains, which have ITS longer than 1 kb, did not allow the amplification of the ITS.
The S-D-Bact-1522-b-S-20/L-D-Bact-132-a-A-18 and 1406F/23Sr primer sets did not allow the amplification of all the spacers of a model community containing five different bacterial species. ARISA profiles obtained with primer set 1406F/23Sr lacked several peaks, while those successfully amplified showed a marked reduction in peak intensity. The S-D-Bact-1522-b-S-20/L-D-Bact-132-a-A-18 primer set generated a lower number of peaks than expected, even though it was able to amplify all the spacers when the template was purified DNA from single strains rather than a mixture of DNA from different species. The S-D-Bact-1522-b-S-20/L-D-Bact-132-a-A-18 set generated profiles that lacked the peaks of P. stutzeri
even when it represented 50% of the community (Fig. ). The ITSF/ITSReub primer set amplified all the strains in the DNA mixture when these DNA were mixed in comparable amounts (Fig. ) but did not amplify the spacer of A. undicula
when it represented less than 5% of the bacterial community DNA, like the S-D-Bact-1522-b-S-20/L-D-Bact-132-a-A-18 primer set (Fig. ). Instead, the spacer of P. stutzeri
was amplified by the ITSF/ITSReub primer set even when it represented only 0.1% of the community. This difference could have been due to different spacer sizes. In fact, the spacer of A. undicula
is about 1,250 bp long, while that of P. stutzeri
is about 530 bp long. The preferential amplification of the shorter templates is a bias of the PCR due to the kinetics of the reaction, possibly determining a false view of the real bacterial community structure. In this respect, the ITSF/ITSReub primer set would be more suitable than the other two primer sets because it reduces to about 120 the numbers of base pairs of 16S and 23S rRNA genes amplified by the PCR, while the S-D-Bact-1522-b-S-20/L-D-Bact-132-a-A-18 and 1406F/23Sr primer sets, respectively, amplify about 185 and 320 bp of the 16S and 23S rRNA genes (Fig. ). The amplification of shorter stretches of the 16S and 23S rRNA genes would optimize the resolution power of the Gene Scan software, which tends to merge peaks with sizes over 500 bp, as observed by Suzuki et al. (24
). Similar considerations could be made for the ARISA of S. meliloti
that is known to have an ITS longer than 1 kb and did not yield any peak with primer set 1406F/23Sr, despite no primer mismatches were found with the 16S and 23S rRNA gene sequences.
The relative peak fluorescences obtained by the ITSF/ITSReub primer set for the different strains in the mixture when different amounts of total DNA were used in the PCR (Fig. ) showed a good correspondence with the expected relative fluorescences calculated on the basis of the amount of DNA of each species added in the reaction, corrected for the factors genome size and ribosomal operon copy number. This indicated that with the DNA mixture used, the ITSF/ITSReub primer set did not suffer PCR biases like those due to substrate reannealing (23
), known to potentially occur during the amplification of a complex DNA mixture. This was not the case for primer sets S-D-Bact-1522-b-S-20/L-D-Bact-132-a-A-18 and 1406F/23Sr, which yielded preferential amplification of S. bovis
and E. coli
A higher number of peaks was found in the ARISA electropherograms of environmental DNA by using the primer set ITSF/ITSReub than with the other two primer sets (Table ); this indicates a more informative capacity of this primer set with respect to the others. No peaks corresponding to spacers above 870 and 767 bp were observed in the environmental community profiles obtained by the S-D-Bact-1522-b-S-20/L-D-Bact-132-a-A-18 and 1406F/23Sr primer sets, respectively. From the same environmental samples, using primer set ITSF/ITSReub, we obtained peaks corresponding to spacers up to 1,341 bp (Table ). Ranjard et al. (19
) analyzed a soil bacterial community with the primer set S-D-Bact-1522-b-S-20/L-D-Bact-132-a-A-18 and observed peaks corresponding to spacers no longer than 800 bp, while Fisher and Triplett (7
), using primer set 1406F/23Sr for the ARISA of freshwater samples, observed peaks up to 1,147 bp, corresponding to spacers of 826 bp. We speculate that these results were due to the preferential amplification of the shorter templates, as suggested by Fisher and Triplett (7
), rather than to the biological absence of species possessing longer spacers (e.g., the α-Proteobacteria
The 1406F/23Sr primer set generated the less-complex profiles, with no peak in many ARISA profiles, especially from soil samples (Table ). Fisher and Triplett standardized ARISA with this primer set by using 150 to 300 ng of DNA as the template (7
). This amount is relatively large compared to what can be obtained by direct extraction from particular samples, such as monuments, artworks, sandy soils, etc. The ARISA results on the model communities allow us to argue that the 1406F/23Sr primer set does not efficiently amplify small amounts of a template, particularly when it consists of soil DNA. However, even when we set up ARISA using large amounts of template (about 300 ng of DNA) the peak profiles were not comparable with the real DNA composition of the model communities (Fig. ).
The ITSF/ITSReub primer set allowed the amplification of only 27 and 22 bp of the 16S and 23S rRNA genes (calculated on the basis of the 16S and 23S rRNA gene sequences of E. coli
O157:H7, excluding the primer sequences) and hence cannot be used for the taxonomic identification of the species by sequencing ARISA fragments and aligning the 16S or 23S rRNA gene stretches in the electronic database. When the principal research purpose includes the identification of species represented by ARISA fragments, the S-D-Bact-1522-b-S-20/L-D-Bact-132-a-A-18 set, which amplifies 115 bp of the 23S rRNA gene, or the 1406F/23Sr primer set, which amplifies 206 and 115 bp of the 16S and 23S rRNA gene, respectively (Fig. ), would be more suitable. However, a specific database for the bacterial ITS, the Ribosomal Internal Spacer Sequence Collection (RISSC) database (9
) is now available, and the number of sequences deposited (2,146 at the end of May 2003) is constantly increasing. This sequence database tool could be used for the taxonomic identification of ARISA fragment sequences independently of the 16S or 23S rRNA gene stretches.
The different efficiencies among the three tested primer sets in ARISA profiling of complex bacterial communities can be partially explained by the comparison of the primer sequences with the 16S and 23S rRNA gene database. All the primers were found to have some mismatches with some sequences in the database (Table ). However, primers S-D-Bact-1522-b-S-20, L-D-Bact-132-a-A-18, and 23Sr did not match with four, one, and one bacterial division, respectively. Moreover the lowest percentages of sequences among those examined having mismatches at the 3′ end of the primer sequence, 0.1 and 0.18%, were found for primers ITSF and ITSReub, respectively. The in silico analysis of melting temperatures and putative secondary structures further indicated the ITSF/ITSReub primer set as the most effective in binding the target sequences. These observations could explain the different efficiencies of the three primer sets in amplifying the environmental samples and demonstrate the intrinsic difficulty in obtaining a primer set really universal for the bacterial domain.
This work presents a first tentative identification of a primer set suitable to reduce PCR biases (such as selective amplification of some templates in a mixture of DNA) during ARISA of complex bacterial communities. Our results have shown that the ITSF/ITSReub primer set emphasizes the capacity of ARISA to powerfully explore microbial diversity and to create complex, easy-to-analyze molecular fingerprintings. We observed that the ITSF/ITSReub primer set does not lead to a total avoidance of PCR biases, but it does reduce the effects of such bias in ARISA, allowing us to obtain a more reliable global view of communities than the other primer sets (Fig. ). Hence, we suggest that the use of the ITSF/ITSReub primer set is appropriate for research where the purpose is to evaluate bacterial community structure by ARISA.