Polymerase chain reaction primers for highly selective detection of algicidal Proteobacteria
Polymerase chain reaction primers for highly selective detection of algicidal Proteobacteria
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用于高选择性检测杀藻变形菌的聚合酶链反应引物
DOI：
10.1046/j.1444-2906.2001.00253.x
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发表时间：
2001
影响因子：
1.9
通讯作者：
I. Imai
中科院分区：
文献类型：
--
作者：
R. Kondo;I. Imai
菌株R的第122-142(R1)位，S和K的209-230(S1和K2)位，D的210-231(D1)位，S和K的457-478(S2和K1B)位，D的645-664(D2)位和R的839-860(R5B)位。目前在公共数据库中存储的一些杀藻细菌的K1B、D1和D2区序列是相同的。其中一个菌株E401与菌株D的DNA关联度超过70%，证实了它们的同义词。10个同源性搜索未发现细菌16S rDNA中其他位置的序列。因此，我们选择这些位置作为特异序列，利用美国马里兰州罗克维尔的美国典型培养库(ATCC)和日本东京大学应用微生物研究所(IAM)获得的变形杆菌的伽马3亚类，选择性地检测每个细菌菌株。根据16S rDNA的特异序列设计的聚合酶链式反应引物，在上述条件下，通过聚合酶链式反应扩增检测杀藻菌株，但K菌株特异性引物的退火温度为50℃，琼脂糖凝胶电泳法检测。使用这套细菌通用8F和1512R引物对选定的蛋白杆菌菌株的16S rDNA进行PCR扩增，从所有受试菌株中都产生了约1.5kbp的单一条带，但使用S1(5‘-TAGCTCACGCCGAAAGAGAGA-3’)和S2(5‘-CAGCTGCAAGTATTAACTTAC-3’)、K2(5‘-AGGCTGCTGCGGCAAAAAAG-3’)和K1B(5‘-GTTGGCAAAAAG-3’)、K2(5‘-AGGCTGCGGCAAAAAG-3’)和K1B(5‘-GTTGGCAAAAAG-3’)、K2(5‘-AGGCTGCTGCGGCAAAAAG-3’)和K1B(5‘-GTTGGGCAAAATACACCAACACACCC-3’)、K2(5‘-AGGCTGCTGCGGCAAAAAG-3’)和K1B(5‘-GTTGGGCAAAATACACCAACACCC-3’)，1(5‘-GGCTCGCTAGAAGAAG-3’)和D2(5‘-TCTAGATGACTAGTCTGA-3’)引物，R1(5‘-CTTGGGAATCTGCCCGAAGGA-3’)和R5B(5‘-CTTCGCAAAACCGTCTTACG-3’)引物(图2)。
Detection of algicidal bacteria FISHERIES SCIENCE 365 strain, corresponding to Escherichia coli sequence positions 122–142 (R1) for strain R, 209–230 (S1 and K2) for strains S and K, 210–231 (D1) for strain D, 457–478 (S2 and K1B) for strains S and K, 645–664 (D2) for strain D, and 839–860 (R5B) for strain R. Primer specificities with reference to published sequences were checked with the program9 at the DNA Data Bank of Japan, Shizuoka. There were identical sequences of K1B, D1 and D2 regions in some algicidal bacteria currently deposited in public databases. One of these algicidal bacteria, strain E401, shared high levels of DNA relatedness to the strain D exceeding 70% of the DNA–DNA reassociation value, confirming their synonymy. 10 Homology searching for other positions identified no sequence in bacterial 16S rDNA. We therefore chose these positions as specific sequences of PCR primers to selectively detect each bacterial strain using the closely related gamma 3 subclass of the Proteobacteria, which were obtained from American Type Culture Collection (ATCC), Rockville, MD, USA and the Institute of Applied Microbiology (IAM), University of Tokyo, Tokyo, Japan.The specificity of the PCR primers designed from the specific sequence of 16S rDNA was tested for detecting the algicidal strains by means of PCR amplification under the conditions described above, except that the annealing temperature for K strain-specific primers was 50 C, and agarose gel electrophoresis was performed with 2% gel. The PCR amplification of the 16S rDNA of the selected Proteobacterial strains using the set of bacterial universal 8F and 1512R primers resulted in the production of a single band of about 1.5 kbp from all strains tested, but resulted in specific products only, which corresponded to the predicted size of the partial 16S rDNA of each algicidal strain, from the strains S, K, D, and R with the set of S1 (5′-TAGCTCACGCCGAAAGAGAA-3′) and S2 (5′-CAGCTGCAAGTTATTAACTTAC-3′) primers, K2 (5′-AGGCTCTGGCGCAAAGAAAG-3′) and K1B (5′-GTTGGGTATTAACCAACAACCC-3′) primers, D1 (5′-GGCTCTCGCTAGAAGAGAAG-3′) and D2 (5′-TCTAGATGACTCTAGTCTGA-3′) primers, and R1 (5′-CTTGGGAATCTGCCCGAAGGA-3′) and R5B (5′-CTTCGCAAAACCGTTCTTACG-3′) primers, respectively (Fig. 2).