The polymerase chain reaction, or PCR, plays a major role both in the diagnosis of infectious diseases and in research. Since the onset of the COVID-19 pandemic, the term has become widely known. At MEDICA 2022, the BLINK AG from Jena, Germany, presented the BLINK Beads, a technology that is bound to revolutionize the applications of PCR.
Dr. Hartmut Bocker
In this MEDICA-tradefair.com interview, Dr. Hartmut Bocker talks about the BLINK beads and their possible applications and explains how they can support and accelerate digital PCR – also when it comes to the point-of-care.
Dr. Bocker, what type of technology is behind BLINK DX and BLINK Beads?
Dr. Hartmut Bocker: We developed a novel technology for the detection of nucleic acids in diagnostics and R&D. Our BLINK beads are at the core of this advancement. They integrate sample preparation and detection reaction, enable a highly sensitive and precise digital PCR, facilitate high-grade multiplexing and rapid PCR testing in the single-digit minute range. To leverage these advantages, we created instrument platforms for the scientific laboratory and for decentralized in-vitro diagnostics at the point-of-care.
What does digital PCR mean?
Bocker: "Digital" refers to thousands of separate volumes that comprise many parallel reactions. Dependent upon the presence of analytes and the specific primers, the result of these reactions is either negative or positive – their result is "zero" or "one", as it were.
Conventional PCR checks the presence of an analyte in a volume. If it is present, the overall reaction becomes positive. If you want to quantify, you will need a real-time PCR to determine at which cycle the reaction became positive, and you require a standard curve as a reference to calculate the initial concentration of an analyte. With digital PCR, absolute values are obtained by evaluating thousands of parallel reactions – without the need for a reference standard.
What are the advantages of digital PCR?
Bocker: Unlike quantitative real-time PCR (qRT-PCR), digital PCR (dPCR) is far more robust. In the latter, inhibitors play only a minor role because it does not matter by design when the dPCR reaction becomes positive, whether that's after 20 or 30 cycles of PCR. We just look at the result after 45 cycles to determine how many reactions are positive, and how many are negative. If you apply this proportion and use the so-called Poisson distribution formula, you can calculate how much of the respective analyte - for example a virus - was present at the beginning in the sample.
This method is well-suited to find the proverbial "needle in a haystack". It is better than qRT-PCR at illustrating minor differences between analytes, as they are required for copy number aberrations or gene expressions. Similarly, you can detect traces of pathogens, simply because there are so many parallel reactions. In a PCR, where the reaction takes place in a large volume, these few copies would simply disappear, masked by non-specific reactions. QRT-PCR has the edge over dPCR at high analyte concentrations. That is why we developed a way in which the beads can be detected in each cycle and evaluated like a qRT-PCR if the dPCR is not sufficient for an application. This facilitates melting curve analysis.
In summary, digital PCR is more sensitive and enables the detection of molecules in a lower concentration. In our case, the process is much faster at 10-15 minutes per PCR.
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Bocker: They fulfill multiple tasks. The beads simplify sample preparation by amplifying nucleic acids. They can contain coding information thanks to colored particles, can be equipped with specific primers and ultimately form the isolated reaction compartments for the PCR in the ongoing process.
Our beads measure about 100 micrometers in size and comprise a hydrogel, meaning a spongy structure consisting of around 99 percent water. The surface of the gel is chemically functionalized, allowing DNA or RNA to bind reversibly regardless of the sequence – even directly from a lysed sample. In a prior step, users can also bind primer molecules to the beads. These define what the PCR reaction is looking for by each primer pair detecting different DNA or RNA sequences. If you bind different primers to beads that are each coded differently and then bring these groups of beads together, it creates a so-called library with which a multitude of detection reactions can be carried out simultaneously in one sample. The magnetic particles that are embedded in the beads facilitate sample processing with conventional magnetic racks.
During a liquid biopsy, fragments of cell-free DNA, cfDNA, can be captured in blood plasma. These can comprise markers in prenatal screening or for diseases such as cancer. CfDNA nucleic acids accumulate on the surface of the beads, which can then be extracted via magnetism and subsequently used for the PCR. This means that the beads can also be used in sample preparation. For the PCR itself, the beads are then isolated by placing them in an oil. Since the beads are aqueous, they remain spatially isolated in the oil. Hence the individual reactions remain separated, meaning analytes or reagents can no longer be exchanged between beads.
We believe this sample preparation, this integrated workflow, to be the unique selling point: the analytes are enriched with the beads. Very little is lost because the beads are transported as a unit from the preparation to the analysis. Combined with color-coded multiplexing, this saves time and money.
What is a practical example of where the beads can press these advantages?
Bocker: At times during the COVID-19 pandemic, PCR resources had to be conserved. That meant pool testing was used where several samples were combined. If the pool returned positive results, each specimen in the pool had to be retested individually. The beads would allow us to test the samples together. You could assign a color to each individual sample and thus easily find out which sample is positive without having to repeat the test.
In theory, are the beads also suited for point-of-care diagnostics?
Bocker: Yes, they are. For now, we are focusing on research and development in the life sciences. Our device for this is the BLINK X. Laboratories can use it to get to know the beads, improve the success of assay transfers, and optimize or newly develop them. The workflow in this setting still necessitates manual work steps. However, we are targeting increased automation and high-throughput analysis.
We also plan to launch the BLINK One, a point-of-care device for in-vitro diagnostics. Assays developed on the Blink X can be transferred to this platform with minor adjustments. A prepared cartridge will contain the beads in freeze-dried form along with the other reagents. This allows us to carry out the entire detection process, from sample lysis, reaction preparation, rapidPCR (taking less than ten minutes) and data evaluation with the device fully automatically in about 30 to 40 minutes. Other systems require two to three hours just for the digital PCR alone. Later this year, we will present the BLINK One to the public.
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