Q-PCR

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Quantitative Polymerase Chain Reaction (Q-PCR), also called real-time polymerase chain reaction (RT-PCR) is giving insight into the identity and number of the organisms in a microbial consortium.

By applying the method to a process at different time points or when different process parameters are applied, it is possible to study the dynamics of the process. By coupling the numbers with knowledge of the physiological traits of an organism it may be possible to optimize a process, for instance by reducing lag phases, increasing the relative abundance of a certain species or using a species as an indicator of a stable or unstable process.

Contents

Principle

Q-PCR is a PCR-based molecular detection tool able to quantify the amount of a target (gene/organism of interest) in a sample. The method is very quick (1.5-2 hours) and relatively cheap. By targeting specific or more general sequences in the DNA of an organism through the development of primers, the method can be used for the detection of a single species (e.g. Leptospirillum ferrooxidans) up to the detection of, for instance, all Eubacteria or Archaea in a sample. The method is based on the amplification of the target of interest - similar to the PCR reaction. The product is, however, not visualized after the complete reaction but already during the amplification process. This can be done in two ways:

  1. by the binding of a fluorescent dye directly to the double-stranded DNA amplification product;
  2. by the use of a third oligonucleotide (TaqMan probe) in the reaction that contains a fluorescent dye as well as a quencher. If the sequence of this oligonucleotide corresponds to the target it is incorporated in the PCR product and the bond between fluorescent dye and quencher is broken. These results in the formation of a fluorescent signal proportional to the amount of product formed.

The amount of amplification cycles it takes to reach a certain threshold setting (ct-value) can be related to the amount of target material in the sample, making quantification possible.

Advantages

  • Very good quantitative tool;
  • Very sensitive, detects 10 cells per sample or more;
  • High specificity;
  • Fast;
  • Easy to develop high throughput system.

Disadvantages

  • Limited number of targets in the same reaction possible;
  • Sensitive to disturbing substances, requires well-purified DNA;
  • Not suitable for unknown targets/organisms.

Current use in bioleaching studies

Q-PCR is currently widely used in many different research fields for the quantification of micro-organisms. The use in the bioleaching research area is very limited. One example of the comparison of Q-PCR to other molecular techniques like CARD-FISH and plating techniques shows that quantification with Q-PCR is possible in mine waste tailing samples (Schippers, 2005).

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