Cardinale Massimiliano, Brusetti Lorenzo, Quatrini Paola, Borin Sara, Puglia Anna Maria, Rizzi Aurora, Zanardini Elisabetta, Sorlini Claudia, Corselli Cesare, Daffonchio Daniele
Dipartimento di Biologia Cellulare e dello Sviluppo, Sezione di Genetica, Università degli Studi di Palermo, Palermo, Italy.
Appl Environ Microbiol. 2004 Oct;70(10):6147-56. doi: 10.1128/AEM.70.10.6147-6156.2004.
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.
ITSF和ITSReub是为扩增16S - 23S rRNA基因间转录间隔区而设计的一组新引物，已与由1406F和23Sr（M. M. Fisher和E. W. Triplett，《应用与环境微生物学》65:4630 - 4636，1999）以及S - D - Bact - 1522 - b - S - 20和L - D - Bact - 132 - a - A - 18（L. Ranjard等人，《应用与环境微生物学》67:4479 - 4487，2001）组成的引物组进行了比较，后一组引物先前被提议用于复杂细菌群落的自动核糖体基因间间隔区分析（ARISA）。使用这三组引物对农业土壤、污染土壤、玉米青贮饲料、山羊奶、来自意大利帕维亚塞尔托萨正面的一小块大理石样本以及地中海深处高盐缺氧盆地的卤水进行了分析。将ARISA图谱中的峰数、峰大小范围（图谱宽度）和结果的可重复性用作指标来评估这三组引物的效率。总体数据表明，ITSF和ITSReub产生的图谱信息最丰富（就峰数而言）且可重复性最高，并且与其他引物组相比，能产生更宽范围的间隔区大小（134至1387），其他引物组在检测长片段方面存在局限性。用特定细菌物种的人工混合物评估了DNA模板的最小量和对少量DNA群体的检测灵敏度。ITSF和ITSReub在DNA模板浓度为280至0.14 ng μl⁻¹时能扩增所有细菌，而其他引物组无法检测到一种或多种细菌菌株的间隔区。尽管由ITSF和ITSReub组成的引物组以及由S - D - Bact - 1522 - b - S - 20和L - D - Bact - 132 - a - A - 18组成的引物组对与其他物种DNA混合的豆科根瘤菌DNA显示出相似的灵敏度，但S - D - Bact - 1522 - b - S - 20和L - D - Bact - 132 - a - A - 18引物组无法检测到施氏假单胞菌的DNA。
