Dikhanov G G, Podladchikova O N
Mol Biol (Mosk). 1990 Jul-Aug;24(4):1010-6.
In order to create a rDNA probe for plague agent (Yersinia pestis) double-stranded DNA fragments complementary to 5'-region of 16S rRNA were synthetized with the help of reverse transcriptase. The fragments were cloned into plasmid vector pUC19 in Escherichia coli. To select plasmids with specific for Y. pestis sequences, recombinant clones and plasmids purified from them were cross-hybridized to [gamma-32 P]-labelled 16S rRNA of E. coli and Y. pestis. As was shown after sequencing of recombinant plasmids, those that did not hybridize to 16S rRNA of E. coli carried a DNA copy of variable region V1 of Y. pestis 16S rRNA. This region was used as a basis for the construction of rDNA probe for genus-specific determination of Yersinia.
为了构建针对鼠疫病原体（耶尔森氏菌鼠疫杆菌）的重组DNA探针，借助逆转录酶合成了与16S rRNA 5'区域互补的双链DNA片段。这些片段被克隆到大肠杆菌中的质粒载体pUC19中。为了筛选出具有鼠疫杆菌特异性序列的质粒，将重组克隆以及从它们中纯化出的质粒与用[γ-32P]标记的大肠杆菌和鼠疫杆菌的16S rRNA进行交叉杂交。对重组质粒进行测序后发现，那些不与大肠杆菌的16S rRNA杂交的质粒携带了鼠疫杆菌16S rRNA可变区V1的DNA拷贝。该区域被用作构建用于耶尔森氏菌属特异性鉴定的重组DNA探针的基础。
