Detection and quantification of petroleum hydrocarbon degrading bacteria targeting catechol cleavage genes

为了对能够降解芳香族化合物的细菌群体进行一般性检测，设计了两个PCR引物组，其可以分别从多种儿茶酚1，2-双加氧酶（C12 O）和儿茶酚2，3-双加氧酶（C23 O）基因扩增特异性片段。根据GenBank中11个C12 O基因的同源序列设计C12 O靶向引物，根据17个已知C23 O基因的同源序列设计C23 O靶向引物。还从内部同源区域设计寡核苷酸探针（C12 Op和C23 Op）以鉴定扩增片段。使用已知携带用于引物和探针设计的C12 O和/或C23 O基因的真实细菌菌株证实引物组和探针的特异性。用C12 O引物对6株已知的C12 O携带菌中的5株进行PCR扩增，扩增产物中有4株为阳性信号。 ...更多信息 用C12 Op进行Southern杂交。C23 O引物从用于其设计的所有11种测试的携带C230的细菌菌株中扩增出预期大小的DNA片段，而C23 Op在来自9种菌株的扩增片段中检测到阳性信号。另一方面，阴性对照未扩增出DNA片段。为了评估所设计的引物和探针用于芳香族化合物降解细菌的一般检测的适用性，将它们应用于从各种环境样品中新分离的野生型苯酚和/对苯甲酸酯降解细菌。C120和/或C230引物从106株野生型菌株中的69株中扩增出预期大小的DNA片段，而C120 p和/或C230 p引物在63株菌株的扩增片段中检测到阳性信号。这些结果表明，我们的引物和探针系统可以检测到相当大比例的细菌，可以通过邻苯二酚裂解途径降解芳香族化合物。最后，建立了环境样品中邻苯二酚裂解基因的定量方法。使用MPN-PCR与降压PCR，靶基因成功地定量对各种环境样品。这些技术研究在这项工作中将适用于生物修复。少

For the general detection of bacterial populations capable of degrading aromatic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenasc (C23O) genes. The C12O-targeting primer set (C12O primers) was designed based on the homologous regions of 11 C12O genes listed in the GenBank, while the C23O-targeting one (C23O primers) was designed based on those of 17 known C23O genes. Oligonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions to identify the amplified fragments. The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the C12O and/or C23O genes used for the primer and probe design. PCR with the C12O primers amplified DNA fragments of the expected sizes from 5 of the 6 known C12O-carrying bacterial strains tested, and positive signals were obtained from 4 of the 5 ampli … More fied fragments on Southern hybridization with the C12Op. The C23O primers amplified DNA fragments of the expected size from all the 11 tested C230-carrying bacterial strains used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments were amplified from the negative controls. To evaluate the applicability of the designed primers and probes for the general detection of aromatic compound-degrading bacteria, they were applied to wild-type phenol- and/op benzoate-degrading bacteria newly isolated from a various environment samples. The C12O and/or C23O primers amplified DNA fragments of the expected size from 69 of the 106 wild-type strains tested, while the C120p and/or C23Op detected positive signals in the amplfied fragemnts from 63 strains. These results suggest that our primer and probe systems can detect a considerable proportion of bacteria which can degrade aromatic compounds via catechol cleavage pathways. And finally we also developed the quantifcation method for the genes coding catechol cleavage in the environmental samples. Using MPN-PCR with stepdown PCR, target genes were successfully quantified against various environmental samples. These techniques investigated in this work would be applicable to the bioremediation. Less