Estimation of the abundance of an uncultured soil bacterial strain by a competitive quantitative PCR method
Estimation of the abundance of an uncultured soil bacterial strain by a competitive quantitative PCR method
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DOI：
10.1128/aem.62.10.3787-3793.1996
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发表时间：
1996-10-01
影响因子：
4.4
通讯作者：
Ogram, A
中科院分区：
文献类型：
--
作者：
Lee, SY;Bollinger, J;Ogram, A
菌株EA25是从华盛顿州东部土壤中提取的DNA扩增的细菌16S rRNA基因序列克隆文库中鉴定出来的。EA25随后被证明与植物属和衣原体的成员有关，并与在澳大利亚土壤样品中发现的菌株MC18最接近（93%相似度）（W. Liesack和E. Stackebrandt， J. Bacteriol, 173:5072-5078, 1992）。采用Zachar等人（V. Zachar， R. a . Thomas, and a . S. Goustin，核酸杂志，21:2017-2018,1993）开发的竞争定量PCR方法估计该未培养菌株在土壤中的丰度。EA25的丰度是根据提取的DNA序列的拷贝数和DIVA提取的效率来估计的，此外，添加到土壤中的大肠杆菌DNA的扩增率与大肠杆菌实验室培养的DNA相似，EA25 16S rRNA基因的数量估计为每克土壤2.17 × 10(8)个拷贝。表明与EA25和澳大利亚菌株相似的菌株可能在温带土壤中广泛分布并大量存在，这是首次从土壤中未培养菌株中枚举16S rDNA拷贝。
Strain EA25 was identified in a clone library of bacterial 16S rRNA gene sequences that had been amplified from DNA extracted from soil collected in eastern Washington State. EA25 was subsequently shown to be related to members of the genera Planctomyces and Chlamydia and most closely related (93% similarity) to strain MC18, a strain identified in an Australian soil sample (W. Liesack and E. Stackebrandt, J. Bacteriol, 174:5072-5078, 1992), A competitive quantitative PCR method developed by Zachar et al, (V. Zachar, R. A. Thomas, and A. S. Goustin, Nucleic Acids Res. 21:2017-2018, 1993) was used to estimate the abundance of this uncultured strain in soil, An estimation of the abundance of EA25 was based on the number of copies of the sequence in the DNA extracted and the efficiency of the DIVA extraction, In addition, amplification rates of Escherichia coli DNAs added to soil were shown to be similar to those of DNAs from laboratory cultures of E. coli, The number of EA25 16S rRNA genes was estimated to be 2.17 x 10(8) copies per g of soil, suggesting that strains similar to EA25 and the similar Australian strain could be widely distributed and present in significant numbers in soils from temperate regions, This represents the first enumeration of 16S rDNA copies from an uncultured strain in soil.