A novel and efficient assay for identification and quantification of Acidithiobacillus ferrooxidans in bioleaching samples

Acidithiobacillus ferrooxidans is a Gram-negative, acidophilic, and chemolithotrophic bacterium that is active in bioleaching. The leaching efficacy is directly influenced by the biomass changes of this specie in bioleaching microbial community. In order to perform a simple and sensitive assay on A. ferrooxidans from mixed strains in this process, a novel assay was developed based on sandwich hybridization assay with the aid of S1 nuclease treatment and fluorescent labeling. In the work, a designed DNA probe complementary to the conservative region of its 16S rRNA was synthesized, which showed high accuracy for distinguishing homologous species with the exclusion of even-only two base pairs difference. The specificity of this assay was verified in different systems with mixed strains, and the quantitative result was proved by comparison of microscopic cell counting. The detection sensitivity was about 8 x 10(2) cells/ml and the inter-assay coefficient of variation of three independent assays was from 3.8 to 7.7 %, respectively. In addition, the cycle of assay was about 3-4 h when the cost estimated was less than $0.5 per sample. This assay method might be applied for identifying and monitoring any kind of bacterial strain from a mixed microbial flora in bioleaching or other areas.