Quality of ORVIDetect analyses

To perform our ORVIdetect DBA analyses we make use of the qPCR technology, which stands for quantitative Polymerase Chain Reaction. Using the qPCR analysis technology micro-organisms are detected and quantified making use of unique features on their genetic material (DNA). You have probably never performed a qPCR analysis yourself, that’s why we wanted to show you how we do it and which quality controls we include.

Step 1: Designing an analysis
Step 2: Sampling and conservation
Step 3: DNA extraction
Step 4: Preparation of analyses
Step 5: Performing analyses

Step 1: Design a qPCR analysis
We need to have designed an analysis before you can order it. To this end we have developed our own primer design software so we can develop analyses for specific bacterial species, groups and/or reactions (enzymes) in a short timeframe.

After we have developed an analysis it is tested, optimised and validated in our own laboratories. We perform the analysis on different types of samples: one sample where we expect a negative result, one sample where we know the target bacteria are present and one sample that we would expect to receive from our clients (for example groundwater from a contaminated site). The analysis is also verified by an external accredited laboratory: does the detected bacterial species match the species for which the analysis was developed (by DNA sequencing).

Step 2: Sampling and conservation
When you have ordered an ORVIdetect DBA analysis, we will send you special sampling material which allows you to take your own groundwater samples. Our sampling bottles contain a fixative which kills all the bacteria that are present. We can do this because we don’t need to keep the bacteria alive, we detect their DNA (which is kept intact in the fixative) instead of growing/culturing them in the lab. Another important advantage is that you no longer have to cool you samples and you can send them by regular post instead of 24 hour delivery. After we have received your samples we will register them in our database and send you an email to let you know they have arrived.

Step 3: DNA extraction
The next step is to extract and purify all the DNA that is present in the bacteria in your groundwater samples. To do this we first break open the bacteria to release all the DNA (and all the cell components). Then all the unwanted components are removed step by step until only the DNA remains. Generally speaking DNA extraction from more complex samples (groundwater containing a lot of sediment) is less efficient, which means these samples will potentially have a higher detection limit.

We keep the DNA that we have purified out of your samples in our freezers. Because we only use a small amount for the analyses it is possible to perform additional analyses on the same sample later on against reduced costs.

Step 4: Preparing the analysis reactions
After the DNA has been extracted and purified we can perform the analyses. Because the qPCR analysis method is so sensitive it is paramount that no external DNA accidentally enters the reaction, for example from materials used or our own skin. That is why we always take the following precautions:

  • The analyses are prepared in a special UV kabinet. First we degrade all external DNA using UV lights. During the preparation procedures the air is continuously treated with UV light.
  • All materials and chemicals we use are certified DNA and RNA free and are not reused.
  • We always wear disposable gloves and cuffs to prevent contamination from our own skin.

Step 5: Performing the analysis
When all the reaction have been prepared they are placed in the qPCR machine which performs the actual analysis. Orvion uses Roche’s newest qPCR machine, the LightCycler 96, with which 96 reaction can be performed simultaneously in about one hour.

Once finished we first check the quality controls that are always included (see below). If these are good, the quantitative results are generated. The results are sent to you in a digital analysis certificate. Your result: the number of Dehalococcoides bacteria per millilitre groundwater.

Quality controls performed:

  • DNA extraction control (DEC): to determine the efficiency of DNA extraction and purification
  • Inhibition control (IHC): a control to determine whether unwanted compounds are present that inhibit the analysis. In case of inhibition we will perform an additional purification step free of charge. If inhibition still occurs your sample is unfortunately not suitable for qPCR analysis.
  • Positive control: when performing a qPCR analysis we always include a positive control to determine that reagents and machine are working correctly.
  • Calibration curve: to enable exact quantification of specific DNA in your samples
  • Background control: To check for contamination we always include an analysis to which no DNA is added (a ‘no template control’ or NTC), this analysis should never give a result.

If you would like more detailed information on how the qPCR works then we recommend looking on the internet or reading the following literature:

Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., et al. (2009). The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin. Chem. 55, 611–622.

Stephen Bustin (2013). Expert Guides Definitive qPCR: (I) Basic Principles (II) A Practical guide (III) Assay Design (IV) Nucleic Acid Quality Control.

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