一种快速简单的 PCR 方法，可用于鉴定根际细菌群体中属名 Azospirillum 的分离株。
A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria.
机构信息
National Agricultural Research Center for Hokkaido Region (NARCH), Hitsujigaoka 1, Toyohira-ku, Sapporo, Japan.
出版信息
J Appl Microbiol. 2011 Oct;111(4):915-24. doi: 10.1111/j.1365-2672.2011.05115.x. Epub 2011 Sep 8.
AIMS
To develop a rapid and simple genus-specific polymerase chain reaction (PCR) method for detecting and identifying isolates of the genus Azospirillum which is well-recognized as plant growth-promoting rhizobacterium.
METHODS AND RESULTS
Nine pairs of PCR primers were designed based on the Azospirillum 16S rRNA, ipdC, nifA and nifH genes to assess their genus specificity by testing against 12 Azospirillum (from seven species) and 15 non-Azospirillum reference strains, as compared with the fAZO/rAZO pair reported by Baudoin et al. (J Appl Microbiol, 108, 2010, 25). Among the primer pairs assessed, the Az16S-A pair designed on the 16S rRNA gene sequence showed the highest genus specificity: it successfully yielded a single amplicon of the expected size in all the 12 Azospirillum strains and for a close relative, Rhodocista centenaria. The PCR with the Az16S-A primers generated a detectable amount of the amplicon from ≥10³ CFU ml⁻¹ of Azospirillum cell suspensions even in the presence of contaminants and accurately discriminated Azospirillum and non-Azospirillum species in both 35 Azospirillum-like and 70 unknown isolates from plant roots and rhizosphere soils.
CONCLUSIONS
We developed a rapid and simple PCR method for detecting and identifying Azospirillum isolates within populations of rhizosphere bacteria.
SIGNIFICANCE AND IMPACT OF THE STUDY
The method developed would serve as a useful tool for isolating a variety of indigenous Azospirillum bacteria from agricultural samples.
目的
开发一种快速简单的种特异性聚合酶链反应（PCR）方法，用于检测和鉴定被广泛认为是植物促生根际细菌的 Azospirillum 属分离株。
方法和结果
基于 Azospirillum 16S rRNA、ipdC、nifA 和 nifH 基因设计了 9 对 PCR 引物，通过与 12 株（来自 7 个种）Azospirillum 和 15 株非 Azospirillum 参考菌株进行测试，评估其种特异性，与 Baudoin 等人报道的 fAZO/rAZO 对进行比较。（J Appl Microbiol, 108, 2010, 25）。在所评估的引物对中，基于 16S rRNA 基因序列设计的 Az16S-A 对显示出最高的种特异性：它成功地在所有 12 株 Azospirillum 菌株和密切相关的 Rhodocista centenaria 中产生了预期大小的单一扩增子。即使存在污染物，使用 Az16S-A 引物进行的 PCR 也能从≥10³ CFU ml⁻¹的 Azospirillum 细胞悬浮液中检测到可检测量的扩增子，并能准确区分 Azospirillum 和非 Azospirillum 种在来自植物根和根际土壤的 35 株 Azospirillum 样和 70 株未知分离株中。
结论
我们开发了一种快速简单的 PCR 方法，用于检测和鉴定根际细菌种群中的 Azospirillum 分离株。
研究的意义和影响
该方法的开发将成为从农业样品中分离各种土著 Azospirillum 细菌的有用工具。
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