| 摘要 |
The present invention discloses a method to identify and quantify environmental microorganisms useful in biomining processes. These microorganisms are basically 10, belonging to Bacteria: Acidiphilium sp., Leptospirillum sp., Sulfobacillus sp., Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans; and Archaea: Acidianus sp., Ferroplasma sp., Metallosphaera sp., Sulfolobus sp. and Thermoplasma sp. The method comprises performing a two-stage PCR known as nested PCR, where in the first stage, called primary PCR, 16S ribosomal DNA sequences (nucleotides 27 to 1492, with E. coli rDNA numbering) are amplified using universal primers for the Bacteria and Archaea kingdoms. In the second stage, these primary amplicons are used as template in qPCR reactions, called secondary PCR, in which internal universal primers for either Bacteria or Archaea kingdoms, as it corresponds, and specific primers designed in our laboratories for different taxons to be determined are used. The first PCR linearly multiplies 16S sequences from bacteria or archaea, thus increasing template abundance for the secondary PCR keeping the original microorganism proportion of the sample. This gives a higher sensitivity to the process when compared to the case of directly using taxon-specific primers on the sample. With qPCR results and other data obtained from the analyzed sample, the microorganism concentration of each analyzed taxon present in the sample is calculated using a mathematical formula.
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