It takes about an hour to prepare the droplets (including mix preparation), two hours for endpoint PCR and less than two minutes per reaction well for fluorescence reading. Afin d’alléger les files d’attentes, il est conseillé, avant de se rendre au laboratoire, de remplir ce formulaire. However, the application of this technique to the field of non-invasive prenatal diagnosis of aneuploidies has been impaired by statistical issues and time-consuming practical issues. positive PCRs) in order to reliability detect molecules originating from the trisomic chromosome. https://doi.org/10.1371/journal.pone.0155009, Editor: Kelvin Yuen Kwong Chan, Hospital Authority, CHINA, Received: May 14, 2015; Accepted: April 22, 2016; Published: May 11, 2016. A ROC curve was then plotted. En tant qu’acteur de santé et contributeur majeur au dépistage de la COVID en France, Cerballiance vous informe des bénéfices et limites des tests aujourd’hui disponibles. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. Les laboratoires de biologie médicale Eurofins réalisent des tests de dépistage COVID-19 par PCR. Slopes for the concentrations (according to the dilution) were computed as a function of the number of probes used in the tested probe sets, and simultaneous confidence intervals for the slope ratios were computed and compared with those of the reference slope obtained with one probe for each chromosome [13]. We designed a two-color octoplex PCR experiment that overcomes limitations on the amount of DNA by increasing the number of targets and thus increasing the number of positive PCRs. Nevertheless, false-negative HCMV PCR results have been reported in AF samples even under these optimal diagnostic conditions (5, 7, 13). made use of this methylation pattern to perform a methylation-sensitive restriction enzyme digestion and then detect placenta-derived, hypermethylated RASSF1A sequences in maternal plasma. 0000096001 00000 n
Yes 0000135917 00000 n
Digital PCR has already been proposed as an alternative technique for aneuploidy NIPT. Secondly, and despite great technical progress in the field of dPCR (enabling thousands of PCRs to be performed in the same well), the number of available target DNAs is sometimes insufficient. We used a set of three FAM TaqMan® hydrolysis assays for the APP, BRWD1 and RUNX1 genes (Assays identities respectively: Hs01344980_cn, Hs03026207_cn and Hs05550012_cn; Life Technologies, Carlsbad, CA, US) and three VIC TaqMan® hydrolysis assays for the ASTN1, FAF1 and PUM1 genes (Assays identities respectively: Hs05795637_cn, Hs06521574_cn and Hs06604919_cn; Life Technologies, Carlsbad, CA, US). 2) The real time PCR GPCMV developed had sensitivity at 50% and 95% of 200 copies/mL and 2500 copies/mL respectively, with a wide linear ranges up to 106 copies/ mL. startxref
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Yes ddPCR protocol was the same as described in the Materials and Methods section. Then, we analyzed the correlation between the observed ratios and the theoretical ones. For artificial mixtures of trisomic and euploid DNA, we performed local polynomial smoothing of the plot for observed and theoretical chromosome ratios with 95% confidence intervals, and then compared the observed curve with the expected curve. We composed a 20 μL reaction mix using 10 μL of 2X ddPCR Supermix for probes, 4 μL of an equimolar mix of the eight TaqMan® assays and 6 μL of plasma DNA. 0000069596 00000 n
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No, Is the Subject Area "Hydrolysis" applicable to this article? The PCR conditions were as follows: 95°C for 10 min, 40 cycles of 94°C for 30 sec and 60°C for 1 min, and then final extension at 98°C for 10 min. The 0% trisomy 21 sample was generated using a mix of 10% of one preparation of normal DNA and 90% of another preparation of normal DNA. Funding: This work was funded by a grant from the French Agence de la Biomédecine (reference: AOR 2010 AMP, diagnostic prénatal et diagnostic génétique) and the Association Maternité et Médecine de la Reproduction charity. 0000071323 00000 n
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Given that this DNA is characterized by a low concentration of fetal DNA and a mix of maternal and fetal DNA, we created a model of mosaic trisomy by mixing DNA from euploid lymphocytes with DNA from trisomy 21 lymphocytes at very low concentrations and variable proportions of trisomy 21. We tested our multiplex assay with artificial mixtures of 0.5 ng/μL DNA (containing 50%, 25%, 10%, 5% or 0% of trisomy 21 DNA) and analyzed the chromosome ratios obtained for different replicates of the same point. Assistance Publique-Hôpitaux de Paris, Hôpital Cochin, Laboratoire de Biochimie et Génétique Moléculaire, Paris, France, Affiliations Vos résultats seront disponibles par envoi d’un mail sécurisé et toujours dans un […] Lastly, given that the amounts of circulating cell-free fetal DNA in the plasma are very low, a large total number of PCRs have to be performed in order to achieve the requisite number of positive PCRs. 0000069774 00000 n
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The samples were collected between 2002 and 2007 in two prenatal diagnosis centers in Poissy Hospital and in Necker Hospital. All patients gave their written, informed consent to participation in the study. In the upper plot, the box displays the median [25th-75th percentiles] for the distribution; the whiskers indicate the data points no further than 1.5 times the interquartile range from the box; crosses represent the group means; data points are plotted as open circles; n = 17 and 187 sample points for the trisomy 21 and normal group respectively. By targeting several genes on the same chromosome with a multiplex of probes labeled with the same fluorophore, we overcame this limitation by substantially increasing the number of positive droplets for a given amount of input DNA. xref
No, Is the Subject Area "Trisomics" applicable to this article? 0000084880 00000 n
We estimated the number of positive PCRs that had to be obtained to detect a small increase in the number of chromosome 21 molecules (compared with reference chromosome molecules) in cases of trisomy 21 with a low fetal fraction (10% and 5%). Furthermore, those invasive procedures are associated with a 0.2–0.5% risk of induced miscarriages [1]. 0000137555 00000 n
Circulating DNA was extracted using the QIAampTM Circulating Nucleic Acid Kit (QIAGEN, Valencia, CA, USA), according to the manufacturer's instructions and resuspended in a final volume of 100 μl. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women. Screening for trisomy 21 is still mostly based on a risk estimation derived from the measurement of (i) biochemical markers in maternal blood and (ii) first trimester nuchal translucency. n is the total number of PCRs. Because MPS or MBM have already proven their efficiency in this purpose, practical use of ddPCR in clinical setting will probably depend upon technical optimization of the technology and on pricing evolution of MPS or MBM to make it (or not) available to all pregnant women. Département d’Obstétrique et de Gynécologie, Hôpital Cochin-Maternité Port-Royal, Université Paris Descartes, Paris, France, Contributed equally to this work with: 0000139312 00000 n
Vous pouvez joindre directement le laboratoire de votre choix sans avoir recours à notre service téléphonique, encart promotionnel indépendant. In this proof of concept study, we validated the PCR system only on samples from pregnancies with trisomy 21 or normal fetal karyotype. Ratio for trisomy 21 (n = 3) and euploid fetus (n = 17) were respectively at 1.10 and 1.02 (p = 0.007) (S3 Table). The World Health Organization, as well as several other national agencies, are still working on different clinical approaches to implement the most relevant treatment in MERS-CoV infection. INFORMATION COVID - Les tests virologiques (RT-PCR) et antigéniques sont réalisables sans ordonnance et pris en charge intégralement par l’Assurance Maladie. Except for sample 66 with the lowest number of positive PCRs (~2800 far from the 5900 required for distinguishing between trisomic and euploid situations), for three other samples the chromosomal ratio was in favor of a trisomy 21. To date, the two main obstacles to the wider application of dPCR are (i) the technical difficulty of performing several thousands of PCRs, and (ii) the high number of positive PCRs required for statistical robustness. Taken as a whole, these results demonstrate that our probes did not interfere with each other. For more information about PLOS Subject Areas, click Fédération de Génétique, CHI de Poissy St Germain, Poissy, France, Affiliation Les tests PCR sont pris en charge à 100 % par l’Assurance maladie. Up to 12 samples can be run at once. Copyright: © 2016 El Khattabi et al. 0000072107 00000 n
We used the QX100 Droplet Digital PCR system (Bio-Rad Laboratories, Hercules, CA, USA). here. GIG, Faculté des Sciences de la Santé, Université de Versailles Saint-Quentin-en-Yvelines, Montigny le bretonneux, France, Affiliation Le laboratoire est un service polyvalent qui propose une offre complète pour les patients hospitalisés du CHIPS et des structures avec lesquelles il dispose d'une convention ainsi qu'à tout patient externe, qu'il soit consultant d'un médecin du CHIPS ou d'un praticien extérieur. Using chromosome 1 as the reference chromosome we tested some plasma DNA samples (n = 20 among which 3 trisomy 21). Firstly, calculations showed that thousands of positive partitions (the droplets, in ddPCR) were needed to achieve statistical significance with high confidence, meaning that thousands of PCRs need to be processed [15, 16]. A: FAM probes (chromosome 21), B: VIC probes (reference chromosome). Approximately 10,000 to 18,000 water-in-oil droplets were generated per replicate, with an average of around 122,000 total droplets per sample. 0000117437 00000 n
A second run of 2 to 8 replicates was performed for samples with less than 5900 positive PCRs for the reference chromosome. Informations. Hence, researchers worldwide have long been seeking to design a more efficient test. Furthermore, further experiments will be necessary for trisomy 18 screening. 0000086137 00000 n
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L’Assurance maladie tient à jour la liste des laboratoires qui pratiquent le test de dépistage de la Covid19 et explique les modalités particulières à suivre avant, pendant et … 0000093356 00000 n
The extracted DNA was stored below -20°C until processing. In case of an euploid fetus, the theoretical ratio r21/ref is 1. For each set of n probes, we plotted the concentration curve of the series dilution (Fig 1) and calculated the slope. = 1.05. n is the total number of PCRs. 0000092277 00000 n
Pour gagner du temps, téléchargez au préalable le Formulaire de renseignements pour la réalisation d’un examen virologique ou sérologique concernant la COVID-19en laboratoire de biologie médicale (485.5 ko) 5 Maternité. e0155009. broad scope, and wide readership – a perfect fit for your research every time. The first problem has been solved by recent technical breakthroughs in dPCR, such as droplet digital PCR (ddPCR), in which thousands of individual PCRs are performed in the same reaction well [11,12], which simplifies laboratory workflow. Lastly, it is essential to avoid interference by targeting genes that lack homology. The theoretical ratio, depending on the fetal fraction, was calculated as follows: r21/ref = [(n21fetal x p) + (n21maternal x (1-p))] / [(nreffetal x p) + (nrefmaternal x (1-p))], where p is the fetal fraction, n21 is the number of chromosomes 21 from maternal (n21maternal) or fetal (n21fetal) origin, and nref is the number of reference chromosome per diploid genome from maternal or fetal origin. PLOS ONE promises fair, rigorous peer review, 0000069286 00000 n
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This condition is met when kref is 1600 (for a fetal fraction of 10%) or 5900 (for a fetal fraction of 5%). hޔ�K(�Q���~�1㕑�bA����Xx/&&�Y��$$�x�,5�ؘ�&+�. None of the approximately 30,000 droplets was SRY-positive, demonstrating an undetectable fetal fraction. 0000098099 00000 n
Objective NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Chan et al. No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, https://doi.org/10.1371/journal.pone.0155009. Although we could demonstrate in this preliminary pilot study that it was possible to obtain a significantly different chromosome 21/reference chromosome ratio between a group of euploid and trisomic samples after stratification according to the karyotype result (Fig 3), a larger cohort will obviously be paramount in order to determine an optimal threshold (for maximizing sensitivity and minimizing the false positive rate) for prospectively classify the samples. %%EOF
Because it has been demonstrated that all the fetal genome is entirely represented among the circulating DNA [19], the choice of the reference chromosome should not be an issue. Discover a faster, simpler path to publishing in a high-quality journal. 0000078779 00000 n
The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample’s trisomic DNA content is as low as 5%. 0000000016 00000 n
here. HCMV strains display genetic variability in the UL144 region, and the analysis of a potential link between UL144 gene polymorphisms and disease severity has scarcely been studied. <<37BA10EBE3E89847B69D27F53A849998>]/Prev 188862>>
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Le Laboratoire d'analyses médicales - Poissy - Cerballiance vous propose une offre de soins pluridiscplinaire de qualité. This proof of concept study demonstrates that multiplex ddPCR is a promising approach for NIPT for trisomy 21, which can be extended to the detection of other aneuploidies and large copy number variations. 0000074897 00000 n
For each sample, the pregnancy term at blood draw, the fetal karyotype and the results of ddPCR (according to the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines [14]) are shown in S2 Table. Service de Gynécologie Obstétrique, Hôpital de Poissy‐St‐Germain, Poissy, France. https://doi.org/10.1371/journal.pone.0155009.s003, https://doi.org/10.1371/journal.pone.0155009.s004. One year after the occurrence of the first case of infection by the Middle East Respiratory Syndrome coronavirus (MERS-CoV) there is no clear consensus on the best treatment to propose. 2 Laboratoire de Cytogénétique, Hôpital Cochin-Maternité Port-Royal, AP-HP, Paris, France. 0000070641 00000 n
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With this threshold, the ratio of sample 41 is a false negative (fetal karyotype was 47,XY,+21). %PDF-1.4
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Click through the PLOS taxonomy to find articles in your field. François Vialard, Test COVID19 N° unique : 09 70 30 17 00 The median (range) total number of positive PCRs obtained for the reference chromosome was 13583 (1424 to 85214). Attention : pour le test PCR COVID-19, pré-opératoire il faut prendre rendez-vous, obligatoirement 48h avant la date de l’intervention. 0000075671 00000 n
According to French law, all women gave written consent for CMV detection by PCR in their AF. By generating uniform water-in-oil droplets, ddPCR partitions the target sequences into approximately 15,000 individual “nanoreactors” in the same well. The investigators performing the digital PCR experiments were not aware of the karyotype result. The lower the proportion of fetal DNA is, the higher the number of positive PCRs are required [10]. Afin d’alléger les files d’attentes, il est conseillé, avant de se rendre au laboratoire, de remplir ce formulaire. Yes Choisissez le laboratoire dont l’adresse de localisation est la plus pratique. 0000072476 00000 n
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Pour cela, il faut contacter le laboratoire en question. In the present study, we developed a novel ddPCR-based method for the non-invasive prenatal screening of trisomy 21 using DNA from maternal plasma. Laboratoire de Cytogénétique, Hôpital Cochin-Maternité Port-Royal, AP-HP, Paris, France, Affiliation A false negative situation for trisomy 21 due to confined placental trisomy 18 is a priori not possible since these two conditions have never been observed concomitantly. The fluorescence signal was measured and analyzed using a QX100 droplet reader and QuantaSoft software (both from Bio-Rad Laboratories, Inc.). 0000083788 00000 n
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The solid black line corresponds to local polynomial smoothing of the curve with 95% confidence intervals (dashed lines), and the solid gray line represents the expected relationship. The workflow is straightforward because a result can be obtained within a day. 0000143525 00000 n
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Les tests virologiques (RT-PCR) sont réalisables sans ordonnance et pris en charge intégralement par l’Assurance Maladie. According to French law, all women gave written consent for CMV detection by PCR in their AF. 0000143044 00000 n
A three-step centrifugation protocol was set up for plasma recovery, with centrifugation (i) at 800 g for 10 min, (ii) at 1600 g for 10 min and (iii) at 16000 g for 10 min. Citation: El Khattabi LA, Rouillac-Le Sciellour C, Le Tessier D, Luscan A, Coustier A, Porcher R, et al. The serial dilutions contained 2, 1, 0.5 and 0.25 ng/μL of DNA. 0000069160 00000 n
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The total number of molecules from a chromosome of interest being the addition of those from the fetus at a proportion of (p) and those from the mother at a proportion of (1-p). 0000071378 00000 n
Among these 153 samples, 115 tested HCMV DNA negative and 38 tested HCMV DNA positive with our in-house HCMV PCR assay . Given that cell free fetal DNA accounts for a weak proportion of the total cell-free DNA in maternal plasma, one needs to count thousands of molecules (i.e. Among these 153 samples, 115 tested HCMV DNA negative and 38 tested HCMV DNA positive with our in-house HCMV PCR assay . Lastly, the chromosome ratio (based on Poisson statistics) was computed using the results of both runs. The principle of dPCR was first described in 1999 [9]. From a healthcare point of view, an improvement in screening efficiency only makes sense if it can be made available to all pregnant women. NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. The fetal karyotype was normal in 192 cases and revealed trisomy 21 in 21 cases. 4 Hôpital Intercommunal de Poissy-Saint Germain, Maternité. k21 and kref are the number of positive droplets for chromosome 21 and the reference chromosome, respectively, and n is the total number of PCRs (with about 256,000 PCRs for samples with sixteen replicates, which is the maximum number here). trailer
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. For a single-sided test and with a 97.5% confidence interval, n21 is higher than nref when k21 > kref + 1.96*sigma_ref (where sigma_ref = √ (n*kref/n*(1-kref/n)). We first tested our multiplex design on serial dilutions of genomic DNA and artificial mixtures of trisomic and euploid DNA. Eight to sixteen replicates were performed for each sample. We sought to quantify the fetal fraction using a duplex hydrolysis probe assays targeting SRY (FAM) and RNaseP (VIC). It then becomes possible to evaluate the absolute quantification of the input DNA by counting the number of positive PCRs. The area under the ROC curve (AUROC: 0.951 (CI95%: 0.861;>0.999)) and the shape of the curve are characteristic of a very good screening test. We are now developing this step in order to further increase the reliability of our ddPCR assay. Responsible for patient recruitment and consent collection: TQ LM VT. Although k21 and kref follow a binomial distribution, they can be considered as having a Gaussian distribution in the present context. Dépistage Covid-19 par PCR avant un départ vers les DOM-TOM (Guadeloupe, Martinique, Réunion, Guyane...) : vos laboratoires MLab réalisent le dépistage dans les jours précédant le départ. Centre de dépistage covid 19 en Yvelines : Trouver un Centre Dépistage Covid-19 - PCR et Sérologie et prenez rendez-vous avec nos professionnels. Competing interests: The authors have declared that no competing interests exist. 0000140501 00000 n
Yes 0000134228 00000 n
The data analysis is very simple and does not require intensive computation or large storage facilities. In contrast, if NIPT is restricted to at-risk women (because of the high cost of MPS-based technologies), the rate of fetal loss would fall but the screening performance would not improve. Then, we normalized the slopes results by dividing the slopes obtained with 2, 3 and 4 FAM or VIC probes targeting the same chromosome by the slope obtained with a single probe (FAM or VIC respectively) (Table 1). The median (range) age of gestation was 16 (9 to 37) weeks. 0000143477 00000 n
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In the upper plot, the box displays the median [25th-75th percentiles] for the distribution; the whiskers indicate the data points no further than 1.5 times the interquartile range from the box; crosses represent the group means; data points are plotted as open circles (n = 10) and sample points (n = 75) for the trisomy 21 and normal group respectively. 0000139794 00000 n
The human cytomegalovirus (HCMV) UL144 gene is a tumor necrosis factor-like receptor with the potential to affect HCMV virulence. Among the nine samples excluded for insufficient number of positive PCRs, four were trisomic 21 (samples 1, 66, 67 and 76). 0000143362 00000 n
After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Les tests sont effectués par les équipes du laboratoire Eurofins. For nine of the samples in our series (samples 1, 9, 40, 64, 66, 67, 76, 84 and 86)the number of positive PCRs was below 5900 (despite a second run). 0000093893 00000 n
3) CMV infections were observed in 14/18 females in group1 and in 9/9 females in group 2. 0000085416 00000 n
Laboratoire de Cytogénétique, Hôpital Cochin-Maternité Port-Royal, AP-HP, Paris, France, Affiliation The authors thank Pr Michel Vidaud, head of the Molecular Genetics lab at the Cochin Hospital, and Franck Letourneur, team leader of the GENOM’IC plateform of the Cochin Institute, for having made available the ddPCR instruments.
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