April 2017, Digital Assays Part I : Partitioning Statistics and Digital PCR (SLAS Technology)

October 2016,  Three-color crystal digital PCR (Biomelocular Detection and Quantification )

Abstract : Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCRTM, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows threecolor multiplexing, which entails a different approach to data analysis. In the present publication, we
present this innovative workflow, which is both fast and user-friendly, and discuss associated data analysis issue, such as fluorescence spillover compensation and data representation. Lastly, we also present proof-of-concept of this three-color detection system, using a quadriplex assay for the detection of EGFR mutations L858R, L861Q and T790M… Read More

January 2013, Droplet microfluidics driven by gradients of confinement (PNAS)mutations L858R, L861Q and T790M… Read More

December 2018, EGFR C797S, EGFR T790M and EGFR sensitizing mutations in non-small cell lung cancer revealed by 6-color crystal digital PCR (OncoTarget)

Abstract:

Background: Detection of EGFR sensitizing and p.T790M and p.C797S resistance mutations is particularly important for non-small cell lung cancer (NSCLC) patient therapy management. Non-invasive blood-based monitoring of these mutations may pave the way to a fine-tuned personalized treatment. Digital PCR has emerged as an extremely sensitive method to detect rare mutations, however its major limitation is the number of hotspots that can be simultaneously differentiated.
Methods: We developed a 6-color digital PCR assay for the detection and quantification of 19 most prevalent EGFR sensitizing and resistance mutations and
evaluated this assay on 82 tumor and plasma samples from NSLC patients.
Results: Limits of detection (LOD) for the 6-color digital PCR assay were assessed on serial dilutions of DNA standards. We found that the 6-color assay enabled
detection of mutant fractions as low as 1 mutant in 1025 wild-type molecules, depending on the mutation targeted, when assayed in a background of 10 000 wildtype DNA copies. EGFR mutant allelic fraction was also measured on tumor and plasma samples by 6-color digital PCR, and displayed a highly significant correlation with next generation sequencing and 3-color digital PCR. Lastly, the 6-color digital PCR assay was performed on several longitudinal plasma samples from four patients and revealed levels of sensitizing and resistance EGFR mutations that reflected well the course of the disease. Read More

August 2017, Crystal digital droplet PCR for detection and quantification of circulating EGFR sensitizing and resistance mutations in advanced nonsmall cell lung cancer (Plos One)

Abstract : Over the past years, targeted therapies using tyrosine kinase inhibitors (TKI) have led to an increase in progression-free survival and response rate for a subgroup of non-small cell lung cancer (NSCLC) patients harbouring specific gene abnormalities compared with chemotherapy. However long-lasting tumor regression is rarely achieved, due to the development of resistant tumoral subclones, which requires alternative therapeutic approaches. Molecular profile at progressive disease is a challenge for making adaptive treatment decisions. The aim of this study was to monitor EGFR-mutant tumors over time based on the
quantity of mutant DNA circulating in plasma (ctDNA), comparing two different methods, Crystal™ Digital™ PCR and Massive Parallel Sequencing (MPS). In plasma circulating cell free DNA (cfDNA) of 61 advanced NSCLC patients we found an overall correlation of 78% between mutated allelic fraction measured by Crystal Digital PCR and MPS… Read More

May 2020, Generation and characterization of two strains of transgene mice expressing chimeric MiniSOG-MusPrP (Journal of Neuroscience Methods)

Abstract:

Background: Although the presences of scrapie associated fibril in the brain tissues is  ultrastructural hallmark for prion diseases, the exact morphological structure of prion during the progression of the disease is still unclear. The host prion protein (PrP) is encoded by PrP gene (PRNP) locating on the chromosome 20 in human and the chromosome 2 in mouse. Recently, a novel correlative light and electron microscopy with Mini Singlet Oxygen Generator (miniSOG) was generated. MiniSOG, a small protein of 106 amino acids, can absorb blue light and emit green fluorescence that is detectable under the fluorescence microscope. MiniSOG can also partially catalyze the polymerization of DAB to form black stained structures in the presence of osmium tetroxide, which is able to be observed under transmission electron microscope.
Methods: Two kinds of miniSOG-PrP expressing recombinant plasmids were generated. Correlative photooxidation and transmission electron microscope were used to detect these plasmids. The plasmids were microinjected into fertilized FVB/NJ eggs and Tg mice expressing miniSOG-PrP fusion proteins were selected after successive bred with PRNP KO Tg mice.
Results: Those two strains of Tg mice, TgSOG23 and Tg231SOG, developed normally and maintained healthy without detectable abnormality after one-year observation. Western blots and immunohistochemical assays with PrP- and miniSOG-specific antibodies confirmed that the chimeric miniSOG-PrP proteins were expressed in the brain tissues of Tg mice. Digital PCR assays proposed that the copy numbers of the inserted external gene in TgSOG23 and Tg231SOG were 2 and 12, respectively. Read More

November 2019, IP10, KC and M-CSF is Remarkably Increased in the Brains from the Various Strains of Experimental Mice Infected with Different
Scrapie Agents (Virologica Sinica)

Abstract:

Activation of inflammatory cells and upregulations of a number of cytokines in the central nervous system (CNS) of patients with prion diseases are frequently observed. To evaluate the potential changes of some brain cytokines that were rarely addressed during prion infection, the levels of 17 different cytokines in the brain homogenates of mice infected with different scrapie mouse-adapted agents were firstly screened with Luminex assay. Significant upregulations of interferon gamma-induced protein 10 (IP10), keratinocyte chemoattractant (KC) and macrophage colony stimulating factor (M-CSF) were frequently detected in the brain lysates of many strains of scrapie infected mice. The upregulations of those three cytokines in the brains of scrapie infected mice were further validated by the individual specific ELISA and immunohistochemical assay. Increased specific mRNAs of IP10, M-CSF and KC in the brains of scrapie infected mice were also detected by the individual specific qRT-PCRs and IP10-specific digital PCR. Dynamic analyses of the brain samples collected at different time points post infection revealed the time-dependent increases of those three cytokines, particularly IP10 during the incubation period of scrapie infection. In addition, we also found that the levels of IP10 in cerebral spinal fluid (CSF) of 45 sporadic Creutzfeldt–Jakob disease (sCJD) patients were slightly but significantly higher than those of the cases who were excluded the diagnosis of prion diseases. These data give us a better understanding of inflammatory reaction during prion infection and progression of prion disease. Read More

August 2019, Cyprinid herpesvirus 3 Evolves In Vitro through an Assemblage of Haplotypes that Alternatively Become Dominant or Under-Represented (Viruses)

Abstract: Viruses are able to evolve in vitro by mutations after serial passages in cell cultures, which can lead to either a loss, or an increase, of virulence. Cyprinid herpesvirus 3 (CyHV-3), a 295-kb double-stranded DNA virus, is the etiological agent of the koi herpesvirus disease (KHVD). To assess the influence of serial passages, an isolate of CyHV-3 (KHV-T) was passaged 99 times onto common carp brain (CCB) cells, and virus virulence was evaluated during passages through the experimental infections of common carp. After 78 CCB passages, the isolate was much less virulent than the original form. A comparative genomic analysis of these three forms of KHV-T (P0, P78 and P99) revealed a limited number of variations. The largest one was a deletion of 1363 bp in the predicted ORF150, which was detected in P78, but not in P99. This unexpected finding was confirmed by conventional PCR and digital PCR. The results presented here primarily suggest that, CyHV-3 evolves, at least in vitro, through an assemblage of haplotypes that alternatively become dominant or under-represented

Read More

February 2020, Duplex digital droplet PCR for the determination of apricot kernels in marzipan (Euro Food Res Technol)

Abstract:

Background: Detection of EGFR sensitizing and p.T790M and p.C797S resistance mutations is particularly important for non-small cell lung cancer (NSCLC) patient therapy management. Non-invasive blood-based monitoring of these mutations may pave the way to a fine-tuned personalized treatment. Digital PCR has emerged as an extremely sensitive method to detect rare mutations, however its major limitation is the number of hotspots that can be simultaneously differentiated.
Methods: We developed a 6-color digital PCR assay for the detection and quantification of 19 most prevalent EGFR sensitizing and resistance mutations and
evaluated this assay on 82 tumor and plasma samples from NSLC patients.
Results: Limits of detection (LOD) for the 6-color digital PCR assay were assessed on serial dilutions of DNA standards. We found that the 6-color assay enabled
detection of mutant fractions as low as 1 mutant in 1025 wild-type molecules, depending on the mutation targeted, when assayed in a background of 10 000 wildtype DNA copies. EGFR mutant allelic fraction was also measured on tumor and plasma samples by 6-color digital PCR, and displayed a highly significant correlation with next generation sequencing and 3-color digital PCR. Lastly, the 6-color digital PCR assay was performed on several longitudinal plasma samples from four patients and revealed levels of sensitizing and resistance EGFR mutations that reflected well the course of the disease. Read More

May 2019, Digital PCR in Myeloid Malignancies: Ready to Replace Quantitative PCR? (International Journal of Molecular Sciences)

Abstract:

New techniques are on the horizon for the detection of small leukemic clones in both, acute leukemias and myeloproliferative disorders. A promising approach is based on digital polymerase chain reaction (PCR). Digital PCR (dPCR) is a breakthrough technology designed to provide absolute nucleic acid quantification. It is particularly useful to detect a low amount of target and therefore it represents an alternative method for detecting measurable residual disease (MRD). The main advantages are the high precision, the very reliable quantification, the absolute quantification without the need for a standard curve, and the excellent reproducibility. Nowadays the main disadvantages of this strategy are the costs that are still higher than standard qPCR, the lack of standardized methods,
and the limited number of laboratories that are equipped with instruments for dPCR. Several studies describing the possibility and advantages of using digital PCR for the detection of specific leukemic transcripts or mutations have already been published. In this review we summarize the available data on the use of dPCR in acute myeloid leukemia and myeloproliferative disorders. Read More