Logo of the Physikalisch-Technische Bundesanstalt

Biomolecular Measurement Methods for Pathogen Detection

Working Group 8.33

Principle of digital PCR


Pathogens like viruses or bacteria contain genetic material that allows their identification. This can support diagnosis of infectious diseases. Polymerase chain reaction (PCR) can be applied to detect their genetic material specifically. Digital PCR not only allows to detect pathogens. It can also be used to quantify the pathogen concentration. This information can be used for diagnosis and therapy control of certain diseases.

Nucleic acid amplification test (NAAT)

NATTs are sensitive methods to detect infectious agents. The classical method uses the polymerase chain reaction (PCR) to amplify a certain sequence of nucleic acids. The reaction mixture contains a number of components. Among others these are the original DNA with the sequence to be amplified (template) , two primers, and an enzyme (Taq polymerase). The reaction is driven by thermal cycling. Amplification is most commonly measured by fluorescence signals.

Usually the amount of DNA is quantified by calibrating the amplification reaction with a DNA standard (quantitative PCR, qPCR). A distinct advantage of digital PCR (see below) is that no calibrator is required.

PCR can only amplify DNA (e.g. from bacteria). Some viruses contain only RNA (e.g. HIV, SARS-CoV-2 virus). Detection of RNA requires initial transcription to "copy DNA" (reverse transcription). This additional step is frequently performed in a separate reaction. It can also be integrated in the PCR reaction (one-step approach).

Some applications need rapid nucleic acid tests for pathogen detection in routine labs. One potential technology is LAMP (Loop-mediated isothermal AMPlification). This is an isothermal method for DNA amplification. Therefore, it does not need thermocycling. Additionally, LAMP utilises 4 to 6 primers for amplification, resulting in very specific amplification. The reaction scheme is involved. Detection of RNA from pathogens in a one-step approach is possible.

Measurement of concentration by digital PCR

The concept of digital PCR is to distribute the reaction in large number of small reaction partitions of equal size. The sample concentration is chosen to give on average less than one copy of the target sequence per each reaction partition. PCR is used to count positive and negative partitions. The number of positive and negative partitions is counted to determined the concentration. There is always a chance to have more than one copy per reaction partition due to counting statistics. This effect is compensated applying a Poisson correction. This allows to determine the number of copies of the target sequence.

The total volume of all reaction partitions is needed to finally determine the concentration. For some digital PCR instruments this volume is fixed by the mechanical dimensions used in the instrument. Recently the use of droplet in oil suspensions are frequently used for digital PCR. The droplets are generated in microfluidic devices. The size of the droplets can be determined by optical microscopy.

Digital PCR does not need calibration material to determine the concentration of pathogens. Thus, digital PCR is a candidate for a primary reference measurement procedure. In addition, novel pathogens can, therefore, more easily be quantified.

Two nucleic acid sequences can be amplified within a single reaction (duplex assay). These two targets can be used to detect different pathogens. An alternative is to use the second target to detect a mutation. Target detection is based on specific fluorescence signals. In duplex assay, the two probes must be labelled with different fluorophores. Typical combinations are the dyes FAM/HEX or FAM/VIC. The result is a two-dimensional scatter graph in which FAM fluorescence is plotted against HEX fluorescence for each droplet.

The Figure shows an example for SARS-CoV-2. The droplets are clustered into four possible groups: single positive FAM (HEX negative), single positive HEX, double positives (HEX and FAM), and double negatives. The distribution of target sequences in the droplets is random following Poisson statistics.