laboratoire poissy pcr

17 Jan laboratoire poissy pcr

0000139312 00000 n In this respect, the ddPCR assay's cost per sample (between 30 and 40 euros, depending on the number of PCR reactions required to obtain 5900 positive droplets) and workflow match the requirements of a population-wide screening test. Data Availability: Data are from the ADN21 study whose authors may be contacted at Laboratoire de Cytogénétique, Hôpital Cochin-Maternité Port-Royal, AP-HP, Paris, France and freely available in supplemental files. Analyzed the data: LAEK CL JMD FV. Here, we designed a technical solution to this problem and tested it on plasma DNA samples from pregnant women, in order to establish whether our dPCR technique reliably discriminates between euploid samples and trisomic fetuses. However, the sensitivity of HCMV PCR in AF is close to 100% when using a PCR test and appropriate timing for amniocentesis (e.g., after 20 weeks of gestation and at least 6 weeks after maternal infection) . 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. However, our ROC curve demonstrates that our assay already has the characteristics of a very good screening test—far better than the current triple-test strategy. test & dépistage du coronavirus à POISSY. In this prospective study, we recruited pregnant women at high risk of chromosomal abnormalities and for whom invasive prenatal diagnosis was indicated on the basis of abnormal biochemical and/or ultrasound results. LABORATOIRE SYNLAB Site RASPAIL - 74, boulevard Raspail Sérologie de 8h à 12h PCR sur rendez-vous de 14h à 18h Tél :01 45 49 11 12-LABORATOIRE SYNLAB Site SAINT SULPICE - 17, rue Saint Sulpice Sérologie uniquement du lundi au vendredi de 7h30 à 15h et le samedi de 8h à 11h30 Tél :01 43 26 60 45 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. We used the QX100 Droplet Digital PCR system (Bio-Rad Laboratories, Hercules, CA, USA). 0000074897 00000 n Les laboratoires de biologie médicale Eurofins réalisent des tests de dépistage COVID-19 par PCR. Next, we established whether the multiplex assay was able to reliably discriminate between euploid and trisomic fetuses in plasma samples from pregnant women at high risk of chromosomal anomalies (according to biochemical and/or ultrasound screening) for whom a fetal karyotype was available. 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. 0000105002 00000 n 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. The equipment is also relatively affordable, and easy to install and implement in routine laboratory practice. 0000143362 00000 n The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. One or two 4 ml EDTA tubes were collected for each patient, yielding an average of around 3 ml of plasma per patient. 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. Service de Gynécologie-Obstétrique, Hôpital Louis Mourier, Hôpitaux Universitaires Paris-Nord Val de Seine, Colombes, France; Université Paris-Diderot, Paris, France, Affiliation Is the Subject Area "Down syndrome" applicable to this article? 0000140389 00000 n Laboratoire des Sources Saint Germain en Laye. 0000073875 00000 n 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. GIG, Faculté des Sciences de la Santé, Université de Versailles Saint-Quentin-en-Yvelines, Montigny le bretonneux, France, Affiliation 0000071616 00000 n Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. 0000137555 00000 n The distribution of the chromosome ratio in trisomy 21 group was significantly shifted toward higher values, when compared with the euploid group (p << 0.0001) (Fig 3). 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. 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. By this normalization, the slopes ratio should be equal to the number of probes used to target the same chromosome and this was the case with our results (Table 1). We used a set of four FAM TaqMan® hydrolysis assays for the BRWD1, LTN1, NCAM2 and RUNX1 genes (Assays identities respectively: Hs03026207_cn, Hs02872951_cn, Hs05556211_cn, Hs05550012_cn; Life Technologies, Carlsbad, CA, US) to detect chromosome 21. 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). 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). 6 Département d'Otologie. 0000096912 00000 n A receiver operating characteristic (ROC) curve of the chromosome ratio was plotted as a predictor of trisomy 21 (Fig 3). Laboratoire de Cytogénétique, Hôpital Cochin-Maternité Port-Royal, AP-HP, Paris, France, Affiliation 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. For more information about PLOS Subject Areas, click The serial dilutions contained 2, 1, 0.5 and 0.25 ng/μL of DNA. Eight replicates were performed for each sample. Adresse : 3 Rue Basset, 78300 Poissy. 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. λ 21 and λ ref are the estimated mean number of copies per droplet, according to the Poisson distribution. Among the nine samples excluded for insufficient number of positive PCRs, four were trisomic 21 (samples 1, 66, 67 and 76). Ratio for trisomy 21 (n = 3) and euploid fetus (n = 17) were respectively at 1.10 and 1.02 (p = 0.007) (S3 Table). 0000074621 00000 n According to French law, all women gave written consent for CMV detection by PCR in their AF. 0000068856 00000 n 0000003053 00000 n 0000073341 00000 n 0000079168 00000 n 0000075421 00000 n Our PCR optimization experiments showed that a combination of multiplexing and automated partitioning of template DNA can be used for NIPT for trisomy 21. <<37BA10EBE3E89847B69D27F53A849998>]/Prev 188862>> The samples were collected between 2002 and 2007 in two prenatal diagnosis centers in Poissy Hospital and in Necker Hospital. broad scope, and wide readership – a perfect fit for your research every time. 182 0 obj <>stream 0000002746 00000 n 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. Moreover, for a given fluorophore, positive droplets remained clearly distinguishable from negative droplets even when multiplexing the eight probes (see S1 Fig). Furthermore, those invasive procedures are associated with a 0.2–0.5% risk of induced miscarriages [1]. Yes Although k21 and kref follow a binomial distribution, they can be considered as having a Gaussian distribution in the present context. No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, https://doi.org/10.1371/journal.pone.0155009. The study was approved by the local investigational review board (Comité de protection des personnes Ile de France XI) with the study approval reference 12079. 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. 7 Unité de Recherche Clinique, AP-HP, Hôpital Necker-E.M., Paris. De la prévention au dépistage, du diagnostic au suivi des traitements, le laboratoire s'engage à vous accompagner, vous conseiller tout au long de votre parcours de soins. 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. 0000117437 00000 n 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. 1 Laboratoire de Virologie, ... CMV polymerase chain reaction (PCR) ... Poissy Hospital, and Béclère Hospital) from January 2008 through December 2009. 2 Laboratoire de Microbiologie Clinique, Centre national de Réfèrence Cytomegalovirus-Laboratoire associé. Citation: El Khattabi LA, Rouillac-Le Sciellour C, Le Tessier D, Luscan A, Coustier A, Porcher R, et al. 0000106612 00000 n The workflow is straightforward because a result can be obtained within a day. 0000138835 00000 n 0000142852 00000 n 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. 0000098099 00000 n 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. 0000071722 00000 n Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. Furthermore, these results show that the experimental ratios are similar to the theoretical ratios, but nevertheless not the same. 0000003863 00000 n 4 Hôpital Intercommunal de Poissy-Saint Germain, Maternité. 84 99 Les tests PCR sont pris en charge à 100 % par l’Assurance maladie. In the lower plot, the AUROC is given with its 95% confidence interval (AUC: Area Under the Curve). 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]. auprès du laboratoire par téléphone Pas de PCR du 18 juillet au 3er aout_ De IIhOO à 16hOO Du 'undi au vendredi 13hOO à 15hOO jusqu'au 20/07 du 'undi au vendredi de 9h30 à 12h SUR RDV 01 45 OO 30 30 sur RDV tout les jours du 'undi au vendredi entre IOH30 et 11H30 (01 42 28 21 28) Responsible for patient recruitment and consent collection: TQ LM VT. To date, only molecule-counting based technologies relying on massively parallel sequencing (MPS) [3], or microarray-based method (MBM) [4] have yielded a useful screening tool. 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%). 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. 0000075671 00000 n We generated 0.5 ng/μL DNA mixtures containing 50%, 25%, 10%, 5% or 0% of trisomy 21 DNA. 0000070565 00000 n 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]. Negative droplets (grey dots) and positive ones (blue dots for FAM+ only, green dots for VIC+ only and brown for FAM+ and VIC+) are assigned as a function of the FAM and VIC florescence amplitudes. The median (range) age of gestation was 16 (9 to 37) weeks. Jean-Michel Dupont. Blood samples were collected (in EDTA tubes) either before the invasive prenatal diagnosis or at least two weeks afterwards. Among these 153 samples, 115 tested HCMV DNA negative and 38 tested HCMV DNA positive with our in-house HCMV PCR assay . Performed the statistical analysis: RP. 0000072415 00000 n Since the discovery of the existence of circulating cell-free fetal DNA [2], several approaches have been used to deduce the number of fetal chromosomes on the basis of the DNA circulating in maternal plasma (despite the fact that the vast majority of DNA in the sample is maternal). 0000092277 00000 n 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. Pour les patients diagnostiqués à l’hôpital ou avec signes de gravité, ces tests seront réalisés dans les hôpitaux. In a final 20 μL reaction volume, we mixed 10 μL of 2X ddPCR Supermix for probes (Bio-Rad Laboratories, Inc.) with 0.5 μL of each TaqMan® assay (the final primers concentration was 450 nM and the final probes concentration was 125 nM), 2 μL of DNA and water up to 20 μL. Furthermore, further experiments will be necessary for trisomy 18 screening. No, Is the Subject Area "Pregnancy" applicable to this article? The investigators performing the digital PCR experiments were not aware of the karyotype result. 0000004079 00000 n Lastly, from an ethical point of view, this test is compatible with the large-scale screening of a low-risk population of pregnant women because there is no longer any need to access sequence information. 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. No, Is the Subject Area "Trisomics" applicable to this article? This observation argues for systematic estimation of the fetal fraction as a pre-analytical quality check. 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. Attention : pour le test PCR COVID-19, pré-opératoire il faut prendre rendez-vous, obligatoirement 48h avant la date de l’intervention. Relative to conventional real-time PCR techniques, individual PCRs provide greater precision and resolution for detecting small concentration differences. 0000003690 00000 n Yes 0000086137 00000 n The test's efficiency and throughput could be increased further by automating the reaction mix preparation and droplet generation. As of 30/03/2020, 715600 people have been infected with COVID-19 worldwide and 35500 people died, essentially due to respiratory distress syndrome (ARDS) complicated in 25% of the with acute renal failure. 3 Réanimation Néonatale, AP-HP, Hôpital Necker-E.M., Paris. Ces tests sont effectués du lundi au samedi matin, avec ou sans ordonnance et sont intégralement pris en charge par la sécurité sociale. 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. Le Test RT-PCR Les tests virologiques RT-PCR étaient uniquement réservés aux personnes présentant des symptômes évocateurs d’une contamination au Covid-19 ou aux sujets contacts . 0000081579 00000 n The chromosome ratio was calculated by dividing the amount of chromosome 21 by that of the reference chromosome. INFORMATION COVID - Les tests virologiques (RT-PCR) et antigéniques sont réalisables sans ordonnance et pris en charge intégralement par l’Assurance Maladie. 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. (2016) Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study. The median (range) total number of positive PCRs obtained for the reference chromosome was 13583 (1424 to 85214). 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. Fan et al. Discover a faster, simpler path to publishing in a high-quality journal. In order to open the field to the screening of both trisomy 21 and 18 at the same time, considering that the trisomy 18 is the second most frequent trisomy sometimes not identified with ultrasound during the 1st or 2nd trimester screening without increasing the final assay price, we designed a set of four VIC TaqMan® hydrolysis assays for the CTIF, RIT2, SMAD4 and TCF4 genes (Assays identities respectively: Hs06500717_cn, Hs06453395_cn, Hs06447834_cn, Hs00372815_cn; Life Technologies) to detect chromosome 18, which served as a reference chromosome. Next, we tested the multiplex PCR assay's sensitivity for detecting small variations in the chromosome ratio in a very-low-concentration sample using artificial mixtures of trisomy 21 DNA and euploid DNA. Nevertheless, false-negative HCMV PCR results have been reported in AF samples even under these optimal diagnostic conditions (5, 7, 13). trailer 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). showed that this approach is precise enough to detect small variation in the chromosome ratio caused by trisomy [15, 16]. 0000093893 00000 n 0000093356 00000 n k 21 and k ref are the number of positive droplets for chromosome 21 and the reference chromosome, respectively. 0000137200 00000 n 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. https://doi.org/10.1371/journal.pone.0155009.s005. However, we validated our multiplex PCR system using chromosome 1 as the reference chromosome and demonstrate that this system is valid whatever the chromosome tested but the ratio threshold will probably differ according to the probes used. On the other hand, three results can be considered as false positive (samples 12, 51 and 145). However, the number of positive PCRs is directly correlated with the number of available target DNA molecules in the sample, which is biologically limited. Yes 0000082668 00000 n 0000106817 00000 n 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)). For more information about PLOS Subject Areas, click 0000143044 00000 n k 21 and k ref are the number of positive droplets for chromosome 21 and the reference chromosome, respectively. https://doi.org/10.1371/journal.pone.0155009.g002. 0000070641 00000 n Yes After performing the ddPCR with our validated octoplex design, we compared the obtained chromosome ratios with the expected ratios. 5 Maternité. 0000143525 00000 n Assistance Publique-Hôpitaux de Paris, Hôpital Cochin, Laboratoire de Biochimie et Génétique Moléculaire, Paris, France, Affiliations It is important to note that the number of positive droplets was correlated with the number of probes, thus indicating an absence of interference. 0000002276 00000 n 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. e0155009. Test COVID19 N° unique : 09 70 30 17 00 Hence, dPCR-based non-invasive prenatal testing (NIPT) for trisomy 21 has not previously been developed. https://doi.org/10.1371/journal.pone.0155009.s006. 0000004044 00000 n 0000096001 00000 n Yes However, to demonstrate that concept works whatever the chromosome chosen as a reference, we also validated our multiplex PCR system using chromosome 1 instead of chromosome 18 as the reference chromosome (S3 Table). 0000143640 00000 n 0000088332 00000 n 0000084880 00000 n 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). 0000143196 00000 n 0000143315 00000 n λ 21 and λ ref are the estimated mean number of copies per droplet, according to the Poisson distribution.

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