New sPCR Technology Runs on Standard Devices

Synergistic PCR (sPCR), a new method of DNA analysis developed for use on a new high-speed assay device, can also be carried out on widely available laboratory real-time PCR (qPCR) instruments and does not require calibration.

While developing a record high-speed genetic research tool, Curiosity Diagnostics (CD; Warsaw, Poland), a spinoff company of the Warsaw Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) and part of the Scope Fluidics group, has developed the new sPCR method, which combines key advantages of the two currently most used methods.

"The DNA assay technique we propose was born during the development of the innovative PCR|ONE analytical instrument, which can be used to test the genetic code in only 7 minutes. This is more than 10-fold shorter time than is required in classic solutions," said Prof. Piotr Garstecki (IPC PAS, CD).

PCR is used both to detect specific DNA fragments and to estimate the original amount of genetic material. In quantitative PCR (qPCR) measurements are usually carried out using real-time PCR – an analogue technique. Due to the sensitivity of PCR to even single particles of impurities, qPCR requires careful, continuous calibration. Conversely, in digital PCR (dPCR) there is no need to calibrate the device, however, because of the need to conduct a large number of reactions in parallel, the testing equipment is expensive and is not as common in laboratories as the analogue apparatus.

sPCR combines the most important advantages of analogue and digital methods to obtain reliable measurements: it is sufficient to dilute a sample into only a dozen or at most several dozen partitions, and calibration is not required.

"A small number of partitions, characteristic of our technique, are of specific practical significance. It means that to perform the analysis all that is needed is the standard well plate format used in popular analogue PCR devices," said Pawel Debski, an IPC PAS PhD student who developed the sPCR method with Curiosity Diagnostics.

Also due to a small number of sample partitions, the sPCR technique is easier to perform and slightly faster than digital variants. Compared to analogue techniques, however, more reagents are required, and so it will not replace the analogue variant. Nevertheless, sPCR could be a valuable addition, as it requires no calibration and so will allow laboratory staff to independently and regularly check the correctness of analogue measurements.

sPCR was developed as an integral component of PCR|ONE, an innovative device designed for rapid DNA analysis. In standard PCR machines, relatively slow heat diffusion between the sample and an adjacent large block of alternately heated or cooled material is used to heat and cool the genetic material. In PCR|ONE, infrared radiation is used to heat the sample rapidly. The diffusion cooling mechanism has also been modified: the block used for this purpose is smaller than in conventional instruments and it is maintained at a constant, strictly controlled temperature. As a result of the technical and analytical improvements, the currently being tested prototypes of PCR|ONE are able to complete DNA assays in less than 15 minutes, and the PCR itself takes only 7 minutes. The first PCR|ONE devices are expected to be commercially available in 2-3 years.

"Our DNA testing technique has been patented. However, we want to emphasize the freedom of using it for non-commercial purposes," said Prof. Garstecki.

The study, by Debski PR et al, was published March 22, 2017, in the journal Scientific Reports.