Limanskiĭ A, Minukhin V, Limanskaia O, Pavlenko N, Mishina M, Tsygenenko A
Zh Mikrobiol Epidemiol Immunobiol. 2005 May-Jun(3):33-9.
Sets of primers for the species-specific detection of P. mirabilis and P. vulgaris by the polymerase chain reaction (PCR) were developed. As targets for these primers beta-lactamase and 16S rRNA gene fragments were chosen on the basis of the multiple leveling of the sequences of the DNA of all known P. mirabilis and P. vulgaris isolates. For differential detection oligonucleotides were selected in such a way that primers, specific for P. vulgaris, contained the non-paired nucleotide for P. mirabilis isolate at the 3'-end, and all other nucleotides were complementary to the beta-lactamase gene fragment. Primers, specific for gene 16S rRNA of P. mirabilis, contained the non-paired nucleotide for P. vulgaris isolates at the 3'-end. Standard PCR was carried out for 6 P. mirabilis and P. vulgaris strains. The use of PCR species-specific primers to P. vulgaris DNA made it possible to amplify the DNA fragment of the expected length only for P. vulgaris isolates, while the result of PCR for P. mirabilis was negative. PCR with primers specific to P. mirabilis permitted the detection of amplicon sized 101 nucleotides pairs only for P. mirabilis strains. These primers were optimized so as to use them in the specific differentiation of closely related P. mirabilis and P. vulgaris species by multiplex PCR. Genus-specific primers permitted the detection of bacterial gyrB gene of the genus Proteus were developed also.
开发了用于通过聚合酶链反应(PCR)对奇异变形杆菌和普通变形杆菌进行种特异性检测的引物组。基于所有已知奇异变形杆菌和普通变形杆菌分离株DNA序列的多重比对,选择β-内酰胺酶和16S rRNA基因片段作为这些引物的靶标。为了进行差异检测,选择寡核苷酸的方式是,对普通变形杆菌特异的引物在3'端含有针对奇异变形杆菌分离株的非配对核苷酸,而所有其他核苷酸与β-内酰胺酶基因片段互补。对奇异变形杆菌16S rRNA基因特异的引物在3'端含有针对普通变形杆菌分离株的非配对核苷酸。对6株奇异变形杆菌和普通变形杆菌菌株进行了标准PCR。使用针对普通变形杆菌DNA的种特异性引物,仅对普通变形杆菌分离株能够扩增出预期长度的DNA片段,而对奇异变形杆菌的PCR结果为阴性。使用针对奇异变形杆菌的引物进行PCR,仅对奇异变形杆菌菌株能够检测到大小为101个核苷酸对的扩增子。对这些引物进行了优化,以便通过多重PCR将其用于密切相关的奇异变形杆菌和普通变形杆菌种的特异性鉴别。还开发了用于检测变形杆菌属细菌gyrB基因的属特异性引物。