Detection of rare events and absolute quantification by PCR
The equipment uses a 3rd generation PCR technique allowing the absolute quantification of DNA targets without a standard range. The sample and its PCR mix are emulsified, in a special oil by a droplet generator, which creates a partitioning of its DNA content and generates several thousand individual PCR bioreactors. It then undergoes a final point PCR (in a standard PCR machine).
Finally, the fluorescence reading of the droplets is done by a reader which successively analyzes the fluorescence of each droplet. The partitioning of the sample allows an increase in sensitivity compared to a conventional qPCR machine. Here we only look if the PCR is positive or negative. The associated software calculates the fraction of the positive partitioning and then estimates the initial target concentration by modeling a distribution according to a Poisson law.
Analytical sensitivity: 0.001%.
Simultaneous analysis of 96 samples possible
Microsatellite instability in colorectal and endometrial cancers
This equipment was used as part of a method validation program for determining microsatellite instability in colorectal and endometrial cancers. In diagnostic routine, this analysis is prescribed for the study of Lynch syndrome and, moreover to guide the prescription of immunotherapy. In this program, the results obtained by ddPCR were compared to those obtained in clinical routine by immunohistochemistry (IHC), by standard PCR. The results obtained by ddPCR are 100% consistent with those of reference techniques (PCR and IHC) and allow the use of ddPCR for diagnostic routine analyses. This work has been published.
EGFR gene mutation in non-small cell lung cancers
The search for EGFR gene mutations is part of the molecular diagnostic assessment of non-small cell lung cancers or non-small cell lung cancer (NSCLC). Our study consisted in validating the performance of ddPCR integrated into a commercial diagnostic kit allowing the detection of EGFR gene mutations involved in sensitivity or resistance to 1st, 2nd or 3rd generation anti-EGFR kinase inhibitors. The analyzes were carried out from DNA extracted from biopsy fragments or circulating free DNA extracted from a blood sample (liquid biopsy). This work was entrusted to a master 2 intern student. The results obtained show that the level of performance of ddPCR applied to the search for EGFR gene mutations is compatible with its use in diagnostic routine. A method validation program is planned.
