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

为了检测能够降解芳香化合物的细菌群体，设计了两组PCR引物，分别可以扩增各种儿茶酚1,2-双加氧酶（c120）和儿茶酚2,3-双加氧asc （C23O）基因的特定片段。C12O靶向引物集（C12O引物）是根据GenBank中列出的11个C12O基因的同源区域设计的，C23O靶向引物集（C23O引物）是根据17个已知C23O基因的同源区域设计的。从内部同源区设计寡核苷酸探针（C12Op和C23Op）对扩增片段进行鉴定。引物组和探针的特异性是用已知携带c120和/或C23O基因的真实菌株来确定的，这些菌株用于引物和探针的设计。用C12O引物对已知的6株携带C12O的菌株进行PCR扩增，其中5株扩增出了预期大小的DNA片段，其中4株与C12Op进行Southern杂交获得了阳性信号。C23O引物从11株携带c230的菌株中扩增出预期大小的DNA片段，而C23Op引物在9株菌株的扩增片段中检测到阳性信号。另一方面，没有从阴性对照中扩增出DNA片段。为了评价所设计的引物和探针对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苯甲酸酯降解细菌。C120p和/或C23O引物扩增了106株野生型菌株中69株的预期大小的DNA片段，而C120p和/或C23Op在扩增的片段中检测到63株的阳性信号。这些结果表明，我们的引物和探针系统可以检测到相当一部分可以通过儿茶酚裂解途径降解芳香族化合物的细菌。最后，我们还建立了环境样品中编码儿茶酚裂解基因的定量方法。利用MPN-PCR和降阶PCR，成功地对不同环境样品的靶基因进行了定量分析。本研究的这些技术将适用于生物修复。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