Wang Gejiao, Gentry Terry J, Grass Gregor, Josephson Karen, Rensing Christopher, Pepper Ian L
Department of Soil, Water and Environmental Science, University of Arizona, Tucson, AZ 85721, USA.
FEMS Microbiol Lett. 2004 Apr 15;233(2):307-14. doi: 10.1016/j.femsle.2004.02.025.
The potential for real-time PCR (RTm-PCR) detection of the genetically engineered strain Pseudomonas putida GN2 was studied during 2-chlorobenzoate (2-CB) degradation in three different soils. The strain contained the constructed plasmid pGN2 which encoded genes for 2-CB oxidation (cbdA) and the green fluorescent protein (gfp). P. putida GN2 numbers were assessed by plating onto 2-CB minimal media and also by RTm-PCR detection of cbdA and gfp. Addition of P. putida GN2 decreased the time required to degrade 2-CB in all tested soils by more than 7 days. The RTm-PCR estimations of P. putida GN2 numbers strongly correlated with those obtained from plate count methods during active 2-CB degradation. However, after 2-CB degradation in the soils had ceased, RTm-PCR estimations of cbdA and gfp genes were generally one order of magnitude lower than those from plate counts. These results indicate the potential for RTm-PCR to rapidly determine degrader numbers in soil following bioaugmentation but also the need to exercise caution when attempting to determine cell numbers of degraders from the RTm-PCR quantification of plasmid encoded genes after substrate is depleted.
研究了在三种不同土壤中2-氯苯甲酸（2-CB）降解过程中，利用实时荧光定量PCR（RTm-PCR）检测基因工程菌株恶臭假单胞菌GN2的可能性。该菌株含有构建的质粒pGN2，其编码2-CB氧化基因（cbdA）和绿色荧光蛋白（gfp）。通过接种到2-CB基本培养基上以及利用RTm-PCR检测cbdA和gfp来评估恶臭假单胞菌GN2的数量。添加恶臭假单胞菌GN2使所有测试土壤中2-CB的降解时间缩短了7天以上。在2-CB活跃降解期间，RTm-PCR对恶臭假单胞菌GN2数量的估计与平板计数法得到的结果高度相关。然而，在土壤中2-CB降解停止后，RTm-PCR对cbdA和gfp基因的估计通常比平板计数法低一个数量级。这些结果表明，RTm-PCR有潜力在生物强化后快速测定土壤中降解菌的数量，但在底物耗尽后试图通过RTm-PCR对质粒编码基因的定量来确定降解菌的细胞数量时，也需要谨慎行事。
