Podladchikova O N, Mishan'kin B N, Dikhanov G G
Mol Gen Mikrobiol Virusol. 1992 Sep-Oct(9-10):21-6.
In order to construct a DNA probe for the plague pathogen detection, we have obtained the recombinant plasmid pRD100 carrying an EcoRI-flanked 140 bp fragment from the genetically silent region of Yersinia pestis species-specific plasmid pYP1. When used as a DNA probe for hybridization of DNA from various strains of 25 bacterial species, this DNA fragment was shown to have the complementary sequences in all investigated Yersinia pestis strains (200), including the plasmid pYP1 lacking ones, and in all the studied Yersinia pseudotuberculosis serotype I strains (80). The search for the probe target in these species has led us to conclusion that it is a specific repeated DNA sequence present in more copies in Yersinia pestis than in Yersinia pseudotuberculosis serotype I. The hybridization of these sequences with the radioactive probe and 24 hours autography makes possible the detection of 1.3 x 10(5) cells of Yersinia pestis and 3 x 10(6) cells of Yersinia pseudotuberculosis serotype I immobilized on the nitrocellulose membranes. Use of the probe for analysis of the nitrocellulose membrane fixed spleen smears from animals that died of experimental plague made possible the detection of Yersinia pestis cells within 48 h.
为构建用于鼠疫病原体检测的DNA探针，我们从鼠疫耶尔森菌种特异性质粒pYP1的基因沉默区获得了携带EcoRI侧翼140 bp片段的重组质粒pRD100。当用作DNA探针与25种细菌的各种菌株的DNA进行杂交时，该DNA片段在所有被研究的鼠疫耶尔森菌菌株（200株）中均显示出互补序列，包括缺乏质粒pYP1的菌株，以及在所有研究的假结核耶尔森菌I型血清型菌株（80株）中也显示出互补序列。在这些物种中寻找探针靶点使我们得出结论，它是一种特异性重复DNA序列，在鼠疫耶尔森菌中的拷贝数比在假结核耶尔森菌I型血清型中更多。这些序列与放射性探针杂交并进行24小时放射自显影，使得能够检测固定在硝酸纤维素膜上的1.3×10⁵个鼠疫耶尔森菌细胞和3×10⁶个假结核耶尔森菌I型血清型细胞。使用该探针分析死于实验性鼠疫的动物的硝酸纤维素膜固定脾脏涂片，使得能够在48小时内检测到鼠疫耶尔森菌细胞。
