Simonet P, Grosjean M C, Misra A K, Nazaret S, Cournoyer B, Normand P
Laboratoire de Microbiologie des Sols, U.R.A. Centre National de la Recherche Scientifique 1450, Université Lyon I, Villeurbanne, France.
Appl Environ Microbiol. 1991 Nov;57(11):3278-86. doi: 10.1128/aem.57.11.3278-3286.1991.
The polymerase chain reaction (PCR) is an in vitro procedure for primer-directed enzymatic amplification of specific template nucleic acid sequences. In order to determine whether a given actinomycete isolated from an actinorhiza (nodule) belongs to the genus Frankia or is a contaminant, we have developed a test based on the PCR. Primers complementary to sequences of two DNA regions corresponding to the nif genes (nifH and nifD) and the rRNA genes (16S and 23S) were specifically chosen to differentially amplify DNAs from Frankia strains but not those from other microorganisms. A series of positive and negative controls were set up by using universal or selective primers resulting in a discriminant amplification, which could be detected after agarose gel electrophoresis. In the nif region, degenerate oligonucleotide primers were used to amplify a target common to all the nitrogen-fixing microorganisms tested, while another set of primers amplified a target with a high specificity for Frankia strains. In the rRNA gene region, universal and specific primers were characterized and tested with DNAs from a wide range of microorganisms. The efficiency of this rapid and sensitive PCR assay was tested with an isolate obtained from Alnus nepalensis nodules, confirming results obtained by nodulation tests.
聚合酶链反应（PCR）是一种用于引物定向酶促扩增特定模板核酸序列的体外方法。为了确定从放线根瘤（根瘤）中分离出的特定放线菌是否属于弗兰克氏菌属或是否为污染物，我们开发了一种基于PCR的检测方法。特意选择了与对应于固氮基因（nifH和nifD）和rRNA基因（16S和23S）的两个DNA区域序列互补的引物，以差异扩增来自弗兰克氏菌菌株的DNA，而不扩增来自其他微生物的DNA。通过使用通用引物或选择性引物建立了一系列阳性和阴性对照，从而实现判别性扩增，可在琼脂糖凝胶电泳后检测到。在nif区域，使用简并寡核苷酸引物扩增所有测试的固氮微生物共有的靶标，而另一组引物扩增对弗兰克氏菌菌株具有高特异性的靶标。在rRNA基因区域，对通用引物和特异性引物进行了表征，并使用来自多种微生物的DNA进行了测试。用从尼泊尔桤木根瘤中获得的分离物对这种快速灵敏的PCR检测方法的效率进行了测试，证实了通过结瘤试验获得的结果。
