Non-isotopic DNA probes for the identification of subgingival microorganisms.
Non-isotopic DNA probes for the identification of subgingival microorganisms.
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用于识别龈下微生物的非同位素 DNA 探针。
DOI：
10.1111/j.1399-302x.1989.tb00405.x
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发表时间：
1989
影响因子：
--
通讯作者：
Smith,CM
中科院分区：
文献类型：
--
作者：
Smith,GL;Socransky,SS;Smith,CM
本研究的目的是检验非同位素DNA探针在主要可培养微生物区系研究中鉴定纯培养物的潜力。用2种方法制备了7种龈下细菌的非同位素DNA探针。首先，通过缺口翻译制备了生物素标记的探针。在第二种方法中，单链DNA通过聚乙烯亚胺与辣根过氧化物酶共价连接。这些探针的相对敏感度和特异度是针对一系列龈下物种的纯培养进行测试的。将等量的肉汤培养物按光学密度标准化，放置在硝酸纤维素或Whatman 541过滤器上，然后处理以裂解细胞，变性并将DNA固定在过滤器上。应用链霉亲和素-碱性磷酸酶检测系统，用生物素标记的同源探针对104-105细胞进行检测。辣根过氧化物酶标记的探针的灵敏度大约低一个数量级。生物素和辣根过氧化物酶标记的探针都显示了与无关物种的非特异性反应，用蛋白酶K和有机溶剂处理过滤器可以消除这些反应。通过比较测试菌株与所涉及的每个交叉反应物种的探针的反应强度，可以区分密切相关物种之间的交叉反应。因此，非同位素DNA探针可用于龈下分离株的快速鉴定。该技术还可用于未知物种菌株的识别和分组。对未知物种的菌株进行探针可以用来快速筛选数百个未知菌株的相关菌株。
The purpose of the present investigation was to examine the potential of non‐isotopic DNA probes to identify pure cultures in predominant cultivable micro‐biota studies. Non‐isotopic DNA probes to 7 subgingival species were prepared by 2 methods. In the first, biotin‐labelled probes were prepared by nick translation. In the second, single‐stranded DNA was covalently linked to horseradish peroxidase via polyethyleneimine. The relative sensitivities and specificities of these probes were tested against pure cultures of a range of subgingival species. Aliquots of broth cultures were standardized by optical densities, placed on nitrocellulose or Whatman 541 filters and then treated to lyse the cells, denature and fix DNA to the filter. Using a streptavidin‐alkaline phosphatase detection system, 104–105cells were detected by homologous biotin‐labelled probes. Horseradish peroxidase‐labelled probes were approximately one order of magnitude less sensitive. Non‐specific reactions with unrelated species, displayed by both biotin‐ and horseradish peroxidase‐labelled probes, were eliminated by treatment of filters with proteinase K and organic solvents. Cross‐reactions between closely related species could be discriminated by comparing reaction intensities of the test strains with probes to the each of the cross‐reacting species involved. Thus, nonisotopic DNA probes could be used for the rapid identification of subgingival isolates. The technique could also be used for the recognition and grouping of strains of unknown species. A probe made to the strain of an unknown species may be used to rapidly screen hundreds of unknown isolates for related strains.