DNA PROBE METHOD FOR THE DETECTION OF SPECIFIC MICROORGANISMS IN THE SOIL BACTERIAL COMMUNITY
DNA PROBE METHOD FOR THE DETECTION OF SPECIFIC MICROORGANISMS IN THE SOIL BACTERIAL COMMUNITY
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DOI：
10.1128/aem.54.3.703-711.1988
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发表时间：
1988-03-01
影响因子：
4.4
通讯作者：
TIEDJE, JM
中科院分区：
文献类型：
--
作者：
HOLBEN, WE;JANSSON, JK;TIEDJE, JM
我们开发了一种从土壤细菌群落中提取纯化细菌DNA的方法。首先将细菌在聚乙烯聚吡咯烷酮存在下从土壤颗粒中分散和分离，聚乙烯聚吡咯烷酮通过吸附到这种不溶性聚合物上来去除腐殖酸污染物。然后通过离心收集土壤细菌，并使用旨在最大限度地破坏存在的各种类型细菌的综合方案进行裂解。通过使用平衡密度梯度从细胞裂解物和剩余的土壤污染物中纯化总细菌DNA。如通过UV光谱分析所确定的，分离的DNA基本上是纯的，至少为48个内切酶长，并且不经历降解，这表明不存在污染性核酸酶活性。分离的DNA容易被外源添加的限制性内切酶消化，并成功地通过狭缝印迹和Southern印迹杂交分析。使用单链，32 P标记的DNA探针，我们可以检测和定量的基础上存在的一个特定的微生物种群在自然土壤群落的DNA序列的存在下，该生物体独特的。我们的方法的灵敏度足以在低至4.3 × 104的密度下检测慢生根瘤菌。104个细胞/g（干重）土壤，这对应于1 μ g DNA样品中约0.2pg的可杂交DNA。
We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded, 32P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 .times. 104 cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1-.mu.g DNA sample.