Liu Chun-Qiang, Plumb Jason, Hendry Philip
CSIRO Molecular and Health Technologies PO Box 184, North Ryde NSW 1670, Australia.
Biotechnol Bioeng. 2006 Jun 5;94(2):330-6. doi: 10.1002/bit.20845.
A SybrGreen real-time PCR assay was developed to detect and quantify both total and selected 16S rDNA species of bacteria and archaea involved in the bioleaching of metals from sulfide ores. A set of specific and universal primers based on 16S rDNA sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of Acidianus brierleyi, Sulfolobus sp., Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus, Acidithiobacillus caldus, and Leptospirillum ferrooxidans. An artificial sequence based on 16S rDNA was constructed to quantify total 16S rDNA in mixed DNA samples. The real-time PCR assay was further validated using a mixture of 16S rDNA amplicons derived from the six different species, each added at a known amount. Finally, the real-time PCR assay was used to monitor the change of 16S rDNA copies of four bioleaching strains inoculated into chalcopyrite airlift column reactors operated at different temperatures. The growth dynamics of these strains correlated well with the expected effects of temperature in the chalcopyrite-leaching environment. The suitability of this method for monitoring microbial populations in industrial bioleaching environments is discussed.
开发了一种SYBR Green实时PCR检测方法，用于检测和定量参与从硫化矿中生物浸出金属的细菌和古菌的总16S rDNA种类和选定的16S rDNA种类。基于16S rDNA序列设计了一组特异性和通用性引物，并对从布氏嗜酸硫化叶菌、硫黄硫化叶菌、嗜热硫化氧化硫杆菌、嗜酸硫杆菌、嗜热嗜酸氧化硫硫杆菌和氧化亚铁钩端螺旋体的代表性菌株中分离的DNA进行特异性检测和定量验证。构建了基于16S rDNA的人工序列，以定量混合DNA样品中的总16S rDNA。使用来自六个不同物种的16S rDNA扩增子混合物（每种以已知量添加）进一步验证了实时PCR检测方法。最后，使用实时PCR检测方法监测接种到在不同温度下运行的黄铜矿气升式柱反应器中的四种生物浸出菌株的16S rDNA拷贝数的变化。这些菌株的生长动态与黄铜矿浸出环境中温度的预期影响密切相关。讨论了该方法在监测工业生物浸出环境中微生物种群方面的适用性。
