Go Bust MIC



What is qPCR?

Unlocking the potential of DNA techniques in biological monitoring entails two pivotal aspects: identification and detection:In both cases, a number of concepts are essential. Yet, navigating this realm requires an understanding of key concepts. Often, DNA concentrations within samples are too minuscule for direct and reliable analysis.  Complicating matters further, the DNA segment of interest constitutes merely a fraction of an organism’s entire genetic makeup, known as the genome.

To overcome these hurdles, DNA undergoes amplification through PCR (Polymerase Chain Reaction), an enzymatic process that replicates small DNA fragments. To amplify a DNA fragment, one uses primers, specially made short syntetic DNA fragments that can start the amplification process. The primers can be chosen to amplify a characteristic piece of DNA from an organism (the DNA barcode).
The term DNA barcoding was proposed by scientists in the early 2000’s as a method for standardised species identification.  The name originated from the fact that a genetic code (a DNA sequence) can be represented in four colours that represent the four building blocks (the nucleotides Adenine, Cytosine, Guanine and Thymine) of DNA, resembling a barcode .

After a PCR, the propagated DNA can be decoded in a process called sequencing. This involves reading the sequence of the propagated DNA. The specific information in the DNA barcode is then used to distinguish and name species. Instead of determining a single DNA barcode from a single organism, the technique can also be applied to a (mixed) sample containing many organisms (DNA metabarcoding). For DNA metabarcoding, there are a number of special sequencing techniques grouped under the term Next-Generation Sequencing (NGS) or High-Throughput Sequencing (HTS). During this process, instead of a single sequence from a single species, it is possible to read up to millions of sequences from multiple species simultaneously. In this way, a lot of genetic information can be obtained from a large number of organisms at once.

A PCR can also be used in MIC analysis to amplify DNA from one specific target species or target function. By making the primers specific enough to fit only the DNA of a single species, it is possible to amplify from a mixed sample of multiple species (such as an MIC sample), only the DNA of this target species. These special primers can be used in quantitative PCR (qPCR). This allows a specific species to be detected in an industrial system, and also allows a statement to be made about differences in the amounts of DNA detected from the particular target species or target function between different sites. The specific qPCR is a very suitable method to test samples specifically for the presence of MIC-related processes. From MICBUSTERS, we offer a ready-to-use solution to perform this on site.


Water filtration to bust MIC-microbiologically induced corrosion
Filtering industrial wastewater to monitor the presence of micro-organisms that create microbiologically induced corrosion

Sampling and sample preservation

Depending on the type of sample, there are several methods for preserving them. Collected industrial water samples, swab samples or corrosion products are best stored with DESS or on a high percentage of ethanol (70-96%). This ethanol can also be used as a source of DNA , as organisms in the ethanol release a lot of DNA.

With the MICBUSTERS kit, DNA from water samples is filtered directly in the field, after which the filter can be kept in a preservation medium until extraction. Alternatively, water can be combined with a preservation medium (such as DESS, RNAlater, Ethanol) to avoid filtration in the field. Storage of non-preserved and/or non-filtered water samples for DNA extraction is always suboptimal, as degradation processes continue even after sampling. Freezing water also has an adverse effect on DNA yield.


DNA extraction involves the release of DNA from the organic and environmental material collected. Filters in which DNA is collected or water samples in which DNA is precipitated contain cell residues in addition to DNA . The same obviously applies to bulk samples that are ground up. During DNA extraction, intact cells are enzymatically and mechanically broken open so that all cell-bound DNA comes into solution. Because DNA contains negatively charged particles, the DNA can be separated from the other components by an electrochemical process, usually by temporarily binding it to a special matrix. At the end of the extraction process, a DNA extract containing pure DNA remains.

We have several types of DNA extraction methods available, including in ready-to-use, MICBUSTERS DNA extraction kit. Different methods each have their advantages and disadvantages, both for maximum DNA yield and extraction efficiency, as well as ease of use, scalability and cost per sample. Consult with our experts to determine the best solution for you.

qPCR testing for MIC in field solution
Field solution to measure for micro-organisms related to MIC corrosion


Normally, DNA concentrations obtained from an extraction are quite low, and the usual techniques for sequence analysis are not sensitive enough to obtain reliable results in a sequence analysis with these very low DNA concentrations. The DNA released from extraction is therefore almost always amplified for further processing. Usually, only the chosen DNA barcode region is amplified. An amplification reaction (the PCR) is an exponential process. That is, the chosen barcode is doubled in each cycle of the reaction (Figure 1). Usually a PCR contains about 25 to 40 cycles, and in an ideal situation, a DNA fragment should thus lead to about a million or a billion copies, respectively.


Data interpretation

The MICBUSTERS qPCR method allows very accurate determination of DNA concentrations of MIC-related species and functional processes in a sample, but to then translate that information into the amount of biomass, or into the number of individuals is not straightforward and it requires the necessary species-specific calibration and validation studies under various conditions.

There are several ways in which the concentration of DNA is affected by external factors within industrial assets. The rate of DNA degradation depends on the species and environmental factors such as temperature, pH, bacterial activity, amount of UV radiation, and amount of organic material (to which DNA can bind), among others.

The measured DNA concentrations can be used to make comparisons in terms of DNA quantities over time or between locations. It is then important to take enough replica samples to make a reliable estimate of the actual concentration, taking into account the spatial variation that is always present in an industrial system.

Nevertheless, direct linkage of measured DNA quantities with numbers or biomass remains not easy. These results serve mainly as relatively easily obtainable information on possible differences in abundances between sites and through time.

Avoidance of Cross-contamination

When working extensively with DNA and handling multiple samples, measures should be taken to prevent cross-contamination between different samples (to avoid false positives). Essential is a separation between so-called pre-PCR (before PCR) and post-PCR (after PCR) activities. PCR products are often relatively small in size (a DNA barcode is only a small piece of DNA) and present in very high concentrations, and can therefore easily contaminate via aerosols the original DNA extracts, which usually contain low concentrations of DNA. To minimise cross-contamination between PCR products, measures can be taken, such as using filter tips for pipetting, so that aerosols cannot enter the pipettes and be transferred to other samples. It is very difficult to completely exclude all sources of possible cross-contamination in advance, and it is therefore essential to include in the laboratory process the necessary control samples that can reveal possible cross-contamination so that this can be corrected if necessary.