Bibliography
Publications
& articles
Read
naica® system 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 PCR™, 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
Oncology
Abstract:
Background and aims
Circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) may be used for diagnostic or prognostic purposes in patients with hepatocellular carcinoma (HCC). We aim to determine whether CTCs or ctDNA are suitable to determine oncogenic mutations in HCC patients.
Methods
Twenty-six mostly advanced HCC patients were enrolled. 30 mL peripheral blood from each patient was obtained. CellSearch system was used for CTC detection. A sequencing panel covering 14 cancer-relevant genes was used to identify oncogenic mutations. TERT promoter C228T and C250T mutations were determined by droplet digital PCR.
Results
CTCs were detected in 27% (7/26) of subjects but at low numbers (median: 2 cells, range: 1–15 cells) and ctDNA in 77% (20/26) of patients. Mutations in ctDNA were identified in several genes: TERT promoter C228T (77%, 20/26), TP53 (23%, 6/26), CTNNB1 (12%, 3/26), PIK3CA (12%, 3/26) and NRAS (4%, 1/26). The TERT C228T mutation was present in all patients with one or more ctDNA mutations, or detectable CTCs. The TERT C228T and TP53 mutations detected in ctDNA were present at higher levels in matched primary HCC tumor tissue. The maximal variant allele frequency (VAF) of ctDNA was linearly correlated with largest tumor size and AFP level (Log10). CtDNA (or TERT C228T) positivity was associated with macrovascular invasion, and positivity of ctDNA (or TERT C228T) or CTCs (≥ 2) correlated with poor patient survival.
Conclusions
Oncogenic mutations could be detected in ctDNA from advanced HCC patients. CtDNA analysis may serve as a promising liquid biopsy to identify druggable mutations.
Abstract:
Circulating tumor DNA (ctDNA) analysis has clinical utility in EGFR mutant NSCLC. Circulating tumor cells (CTCs) consist a unique source of information at the cellular level. Digital PCR (dPCR) is a valuable tool for accurate and valid analysis of gene mutations in liquid biopsy analysis. In the present study we detected EGFR mutations in ctDNA and paired CTCs under osimertinib therapy at two time points using crystal dPCR and the naica® system (Stilla Technologies). We quantified mutation allele frequencies (MAF) of EGFR mutations in 91 plasma cfDNA samples of 48 EGFR mutant NSCLC patients and in 64 matched CTC-derived genomic DNA samples, and the FDA-cleared cobas® EGFR mutation test in 80 identical plasma samples. Direct comparison between crystal dPCR and the cobas EGFR assay revealed a high concordance for all EGFR mutations. Our comparison of crystal dPCR results in ctDNA with the corresponding primary tissue has shown a strong correlation. EGFR mutations analysis in paired CTC-derived gDNA revealed a high heterogeneity. Crystal dPCR offers the unique advantages of high analytical sensitivity, precision, and accuracy for detecting and quantifying multiple EGFR mutations in plasma cfDNA and CTCs of NSCLC patients.
Abstract:
BACKGROUND: Noninvasive plasma-based detection of EGFR mutations using digital PCR promises a fast, sensitive and reliable
approach to predicting the efficiency of EGFR-TKI. However, the low throughput and high cost of digital PCR restricts its clinical
application.
METHODS: We designed a digital PCR assay, which can simultaneously detect 39 mutations of exons 18–21 of the EGFR gene. To
assess overall performance, retrospective FFPE tissues from 30 NSCLC patients and plasma from 33 NSCLC patients were collected
and analysed.
RESULTS: The LoD of the EGFR mutations was as low as 0.308 copies/μL, and the linear correlation between the detected and
expected values at different concentrations (0.01–10%) was low as well. Compared to ARMS-PCR in FFPE, the accuracy values of the
dEGFR39 assay in plasma from 33 patients was 87.88% (29/33, 95% CI 72.67–95.18%). While monitoring the 33 patients, the EGFR
mutation load as assessed by dEGFR39 was associated with the objective response to treatment. Thirteen samples from eight
patients were identified by dEGFR39 to harbour the T790M mutation over time; of these patients, only nine (69%) were detected
using SuperARMS.
CONCLUSION: Our results indicate that dEGFR39 assay is reliable, sensitive and cost-efficient. This method is beneficial for profiling
EGFR mutations for precision therapy and prognosis after TKI treatment, especially in patients with insufficient tissue biopsy
samples.
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
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
Genetics
Abstract:
The quantification of mitochondrial DNA heteroplasmy for the diagnosis of mitochondrial disease or after mitochondrial donation, is performed mainly using next-generation sequencing strategies (NGS). Digital PCR (dPCR) has the potential to offer an accurate alternative for mutation load quantification.
Infectious Diseases
August 2021, Equine Parvovirus-Hepatitis Screening in Horses and Donkeys
with Histopathologic Liver Abnormalities (Viruses)
Abstract
There is strong evidence that equine parvovirus-hepatitis (EqPV-H) is associated with the
onset of Theiler’s disease, an acute hepatic necrosis, in horses. However, the impact of this virus
on other hepatopathies remains unknown. The objective of this retrospective study was to evaluate
the prevalence and quantify the viral loads of EqPV-H in formalin-fixed, paraffin-embedded equine
and donkey livers with various histopathologic abnormalities. The pathologies included cirrhosis,
circulatory disorders of the liver, toxic and metabolic hepatic diseases as well as neoplastic and
inflammatory diseases (n = 84). Eight normal liver samples were included for comparison as controls.
EqPV-H DNA was qualitatively and quantitatively measured by real-time PCR and digital PCR,
respectively. The virus was detected in two livers originating from horses diagnosed with abdominal
neoplasia and liver metastasis (loads of 5 103 and 9.5 103 genome equivalents per million cells).
The amount of viral nucleic acids measured indicates chronic infection or persistence of EqPV-H,
which might have been facilitated by the neoplastic disease. In summary, this study did not provide
evidence for EqPV-H being involved in hepatopathies other than Theiler’s disease.
May 2021, Triplex digital PCR assays for the quantification of intact proviral HIV-1 DNA (Methods)
Abstract
The development of an HIV-1 cure is hampered by the existence of a persistent (latent) reservoir that contains a small proportion of replication-competent intact proviruses which refuels viral replication upon treatment discontinuation. Therefore, an accurate evaluation and quantification of these (intact) proviruses is essential to determine the efficacy of HIV-1 cure strategies which aim to eliminate this reservoir. Here, we present two triplex digital PCR assays which resulted from a combination of two existing methods, the IPDA (a 2-colour digital PCR based method) and Q4PCR assays (4 colour qPCR method), and tested the functionality on a three-colour digital PCR platform. In the present paper, we provide a step-by-step experimental protocol for these triplex digital PCR assays and validate their performance on a latently infected Jurkat cell-line model and HIV-1 patient samples. Our data demonstrates the potential and flexibility of increasing the number of subgenomic regions of HIV-1 within the IPDA to acquire sensitive detection of the HIV-1 reservoir while benefitting from the advantages of a dPCR setup.
February 2021, Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays (EBioMedicine)
Background
Detection of SARS-CoV-2 infections is important for treatment, isolation of infected and exposed individuals, and contact tracing. RT-qPCR is the “gold-standard” method to sensitively detect SARS-CoV-2 RNA, but most laboratory-developed RT-qPCR assays involve complex steps. Here, we aimed to simplify RT-qPCR assays by streamlining reaction setup, eliminating RNA extraction, and proposing reduced-cost detection workflows that avoid the need for expensive qPCR instruments.
Method
A low-cost RT-PCR based “kit” was developed for faster turnaround than the CDC developed protocol. We demonstrated three detection workflows: two that can be deployed in laboratories conducting assays of variable complexity, and one that could be simple enough for point-of-care. Analytical sensitivity was assessed using SARS-CoV-2 RNA spiked in simulated nasal matrix. Clinical performance was evaluated using contrived human nasal matrix (n = 41) and clinical nasal specimens collected from individuals with respiratory symptoms (n = 110).
Finding
The analytical sensitivity of the lyophilised RT-PCR was 10 copies/reaction using purified SARS-CoV-2 RNA, and 20 copies/reaction when using direct lysate in simulated nasal matrix. Evaluation of assay performance on contrived human matrix showed 96.7–100% specificity and 100% sensitivity at ≥20 RNA copies. A head-to-head comparison with the standard CDC protocol on clinical specimens showed 83.8–94.6% sensitivity and 96.8–100% specificity. We found 3.6% indeterminate samples (undetected human control), lower than 8.1% with the standard protocol.
Interpretation
This preliminary work should support laboratories or commercial entities to develop and expand access to Covid-19 testing. Software guidance development for this assay is ongoing to enable implementation in other settings.
Background. Coronavirus disease 2019 (COVID-19) is a global public health problem that has already caused more than 662,000 deaths worldwide. Although the clinical manifestations of COVID-19 are dominated by respiratory symptoms, some patients present other severe damage such as cardiovascular, renal and liver injury or/and multiple organ failure, suggesting a spread of the SARS-CoV-2 in blood. Recent ultrasensitive polymerase chain reaction (PCR) technology now allows absolute quantification of nucleic acids in plasma. We herein intended to use the droplet-based digital PCR technology to obtain sensitive detection and precise quantification of plasma SARS-CoV-2 viral load (SARSCoV-2 RNAaemia) in hospitalized COVID-19 patients.
Methods. Fifty-eight consecutive COVID-19 patients with pneumonia 8 to 12 days after onset of symptoms and 12 healthy controls were analyzed. Disease severity was categorized as mild-to-moderate in 17 patients, severe in 16 patients and critical in 26 patients. Plasma SARS-CoV-2 RNAaemia was quantified by droplet digital Crystal Digital PCR™ nextgeneration technology (Stilla Technologies, Villejuif, France).
Results. Overall, SARS-CoV-2 RNAaemia was detected in 43 (74.1%) patients. Prevalence of positive SARS-CoV-2 RNAaemia correlated with disease severity, ranging from 53% in mild-to-moderate patients to 88% in critically ill patients (p=0.036). Levels of SARS-CoV-2 RNAaemia were associated with severity (p=0.035). Among nine patients who experienced clinical deterioration during follow-up, eight had positive SARS-CoV-2 RNAaemia at baseline while only one critical patient with undetectable SARS-CoV-2 RNAaemia at the time of analysis died at day 27. Conclusion. SARS-CoV-2 RNAaemia measured by droplet-based digital PCR constitutes a promising prognosis biomarker in COVID-19 patients.
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
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
Food and GMO Testing
Abstract:
Breeding exploits novel allelic combinations assured by meiotic recombination. Barley (Hordeum vulgare) single pollen nucleus genotyping enables measurement of meiotic recombination rates in gametes before fertilization without the need for segregating populations. However, so far, established methods rely on whole-genome amplification of every single pollen nucleus due to their limited DNA content, thus restricting the number of analyzed samples. In this study, high-throughput measurements of meiotic recombination rates in barley pollen nuclei without whole-genome amplification were performed through a Crystal Digital PCRTM-based genotyping assay. Meiotic recombination rates within two centromeric and two distal chromosomal intervals were measured in hybrid plants by genotyping a total of >42 000 individual pollen nuclei (up to 4900 nuclei analyzed per plant). Determined recombination frequencies in pollen nuclei were similar to frequencies in segregating populations. We improved the efficiency of the genotyping by pretreating the pollen nuclei with a thermostable restriction enzyme. Additional opportunities for a higher sample throughput and a further increase of the genotyping efficiency are presented and discussed. Taken together, single barley pollen nucleus genotyping based on Crystal Digital PCRTM enables reliable, rapid and high-throughput meiotic recombination measurements within defined chromosomal intervals of intraspecific hybrid plants. The successful encapsulation of nuclei from a range of species with different nuclear and genome sizes suggests that the proposed method is broadly applicable to genotyping single nuclei.
February 2020, Duplex digital droplet PCR for the determination of apricot kernels in marzipan (Euro Food Res & Technol)
Abstract:
Marzipan is a mixture of almonds, sugar, and water. Almonds can be replaced by apricot kernels, which results in a similar product called persipan. Depending on the prices of almonds, proft can be maximized using apricot kernels instead of almonds. If not specifed on the product, this kind of substitution is illegal and is prosecuted by ofcial food control authorities. Likewise, also commercial buyers would like to know which product they bought. Real-time PCR systems for the determination of apricot DNA are already available, however, real-time PCR requires the use of external standards, which have a direct impact on the ccuracy of the measurement. Currently, such standards are not available. In contrast to real-time PCR, digital PCR does not require external standards and exhibits a smaller measurement uncertainty as shown in recent publications. Therefore, we developed a duplex droplet digital PCR system to measure the proportion of apricot in marzipan without the use of external standards. We present validation data and results of an international profciency test, underlining the applicability of this system. Read More
Hematology
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
Pathogen Detection
Abstract:
Modern aquaculture systems are designed for intensive rearing of fish or other species. Both land-based and offshore systems typically contain high loads of biomass and the water quality in these systems is of paramount importance for fish health and production. Microorganisms play a crucial role in removal of organic matter and nitrogen-recycling, production of toxic hydrogen sulfide (H2S), and can affect fish health directly if pathogenic for fish or exerting probiotic properties. Methods currently used in aquaculture for monitoring certain bacteria species numbers still have typically low precision, specificity, sensitivity and are time-consuming. Here, we demonstrate the use of Digital PCR as a powerful tool for absolute quantification of sulfate-reducing bacteria (SRB) and major pathogens in salmon aquaculture, Moritella viscosa, Yersinia ruckeri and Flavobacterium psychrophilum. In addition, an assay for quantification of Listeria monocytogenes, which is a human pathogen bacterium and relevant target associated with salmonid cultivation in recirculating systems and salmon processing, has been assessed. Sudden mass mortality incidents caused by H2S produced by SRB have become of major concern in closed aquaculture systems. An ultra-sensitive assay for quantification of SRB has been established using Desulfovibrio desulfuricans as reference strain. The use of TaqMan® probe technology allowed for the development of multi-plex assays capable of simultaneous quantification of these aquaculture priority bacteria. In single-plex assays, limit of detection was found to be at around 20 fg DNA for M. viscosa, Y. ruckeri and F. psychrophilum, and as low as 2 fg DNA for L. monocytogenes and D. desulfuricans. Read More
Scientific
Resources
Read
MIQE- Minimum Information for Publication of Quantitative Real-time and Digital PCR Experiments
Six-color Crystal Digital PCR™
Scientific
communications
Read
- March 2020, “Crystal Digital PCR™: a fast and high-plex solution for GMO quantification” by Romain Parillaud, Application Specialist at Stilla Technologies. At the 8th Plants & Genomics conference (March 04-05, 2020), Rotterdam, the Netherlands.
- March 2020, “Crystal Digital PCR™ a high plex solution for fast and simultaneous target quantification” by Allison Mallory; Director of Molecular Biology at Stilla Technologies. At TRI-CON Conference (March 01-04, 2020); San Francisco, USA
- March 2020, “Discover the simplicity of Digital PCR” by Alexandra Martin, Application Specialist at Stilla Technologies. At EMBL Virtual Symposium: The Four-Dimension Genome (March 30-31st, 2020).
- April 2020, “Crystal Digital PCR™ For Genome Editing And High Multiplexing Mutation Detection” by Kimberley Gutierrez, Application Specialist at Stilla Technologies. At the Next Gen Omics Series US Digital event (April 7-8, 2020). Thank you to EMBL for their collaboration.
- May 2020, “Crystal Digital PCR™ detection of SARS-CoV-2 using the Naica™ System” by Kimberley Gutierrez, Application Specialist at Stilla Technologies. At Life Science Exhibits Virtual Event (May 7-9, 2020).
- May 2020, “Highly Sensitive Crystal Digital PCR™ Detection Kit for SARS-CoV-2” webinar presented by Romain Parillaud, Application Specialist at Stilla Technologies. In collaboration with Cambridge Healthtech Institute. (May 20, 2020).