Hall Kevin, Miller Charles D, Sorensen Darwin L, Anderson Anne J, Sims Ronald C
North Wind, Inc., 1425 Higham Street, Idaho Falls, ID 83402, USA.
Biodegradation. 2005 Oct;16(5):475-84. doi: 10.1007/s10532-004-5669-y.
A gene probe for the detection of polycyclic aromatic hydrocarbon (PAH) induced nidB and nidA dioxygenase genes has been designed from Mycobacteria JLS, KMS, and MCS. The probe detects a catabolic gene involved in the initial steps of PAH biodegradation in mycobacteria. The gene probe is comprised of three PCR primer sets designed to detect the genes that code for two subunits of the PAH induced dioxygenase enzyme within PAH-degrading mycobacteria. The probe was built by combining three primer sets with a DNA extraction procedure that was designed to lyse the gram-positive mycobacteria cells while in the soil matrix and remove PCR inhibitors. The probe was tested on PAH contaminated soils undergoing bioremediation through landfarming and uncontaminated soils from the same site. The PAH gene probe results demonstrate that the dioxygenase genes can be detected in soils. Sequencing the nidA and nidB PCR products verified that the genes were detected in soil. Comparisons of the sequences obtained from the soil probe to seven known nid gene sequences from various PAH-degrading mycobacteria showed between 97 and 99% nucleotide matches with the nidB gene and 95 and 99% matches with the nidA gene.
已根据分枝杆菌JLS、KMS和MCS设计出一种用于检测多环芳烃（PAH）诱导的nidB和nidA双加氧酶基因的基因探针。该探针可检测分枝杆菌中参与PAH生物降解初始步骤的分解代谢基因。该基因探针由三组PCR引物组成，设计用于检测编码PAH诱导的双加氧酶两个亚基的基因，这些基因存在于降解PAH的分枝杆菌中。该探针通过将三组引物与一种DNA提取程序相结合构建而成，该程序旨在裂解土壤基质中的革兰氏阳性分枝杆菌细胞并去除PCR抑制剂。该探针在通过土地耕作进行生物修复的PAH污染土壤以及同一地点的未污染土壤上进行了测试。PAH基因探针结果表明，双加氧酶基因可在土壤中检测到。对nidA和nidB PCR产物进行测序，证实这些基因在土壤中被检测到。将从土壤探针获得的序列与来自各种降解PAH的分枝杆菌的七个已知nid基因序列进行比较，结果显示与nidB基因的核苷酸匹配率在97%至99%之间，与nidA基因的匹配率在95%至99%之间。
