Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases

Jeddi, Fakhri; Piarroux, Renaud; Mary, Charles

doi:10.1051/parasite/2013051

Cited by 24 publications

(24 citation statements)

References 24 publications

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“…As leukocyte genomic DNA is released into serum during the coagulation [13], and blood clots continuously liberate genomic DNA molecules to this specimen overtime [14], both processes can deliberately be used to increase genomic DNA concentration in serum, which would guarantee the minimal amount of DNA ensuring reliable results. Additionally, some automated DNA extractions designed for serum/plasma co-extracts of PCR inhibitors when whole blood is used (e.g., Nuclisens Easymag System, Biomérieux, Marcy-l'Étoile, France) and additional purification steps are required to achieve amplifiable DNA [15]. These additional steps diminish the high-throughput of JAK2 V617F analysis in a clinical laboratory setting.…”

Section: Introductionmentioning

confidence: 99%

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

et al. 2020

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Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2-∆∆Cq method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2-∆∆Cq method—the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.

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Section: Introductionmentioning

confidence: 99%

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

et al. 2020

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“…Tumor content of > 70%, excluding blood cells, neural cells, and necrosis as much as possible are the basic conditions for accurate molecular diagnosis, especially for analyses by PCR and sequencing. 10 In our case cohort, tissues from 26 cases (8 for biopsy and 18 for craniotomy) were collected with assistance of MR image-guidance during neuronavigational surgery. The most representative tumor sample was selected according to RANO Criteria.…”

Section: Discussionmentioning

confidence: 99%

Methods of Glioma Sample Processing for Molecular Diagnosis for the Glioma Tissue Bank Project

Shi

Aibaidula

Tang

et al. 2015

Biopreservation and Biobanking

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Genome-wide sequencing in glioma samples provides comprehensive insights into oncogenesis and malignant transformation. Several distinct biomarkers have been proven to have clinical significance and are being widely applied in routine clinical practice. Standard sample processing lays the foundation for successful molecular testing. In this study, we found intraoperative neuronavigation ensured higher tumor purity during sample collection, and an automated device helped improve DNA quality and increased yields. These two technologies are beneficial for glioma tissue bank construction and provide for accurate molecular testing during routine clinical practice.

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“…. Genomic DNA was extracted using the NucliSENS TM EasyMAG TM (bioMérieux) system38, eluted in 50 μl and stored at −20 °C. Amplification reactions were performed using a Lightcycler TM 480 (Roche Diagnostics, GmbH, Germany) instrument with Lightcycler TM 480 Probes Master (Roche Diagnostics, GmbH, Germany).…”

Section: Methodsmentioning

confidence: 99%

Opportunistic fungal pathogen Candida glabrata circulates between humans and yellow-legged gulls

Al-Yasiri

Normand

L’Ollivier

et al. 2016

Sci Rep

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The opportunistic pathogenic yeast Candida glabrata is a component of the mycobiota of both humans and yellow-legged gulls that is prone to develop fluconazole resistance. Whether gulls are a reservoir of the yeast and facilitate the dissemination of human C. glabrata strains remains an open question. In this study, MLVA genotyping highlighted the lack of genetic structure of 190 C. glabrata strains isolated from either patients in three hospitals or fecal samples collected from gull breeding colonies located in five distinct areas along the French Mediterranean littoral. Fluconazole-resistant isolates were evenly distributed between both gull and human populations. These findings demonstrate that gulls are a reservoir of this species and facilitate the diffusion of C. glabrata and indirect transmission to human or animal hosts via environmental contamination. This eco-epidemiological view, which can be applied to other vertebrate host species, broadens our perspective regarding the reservoirs and dissemination patterns of antifungal-resistant human pathogenic yeast.

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Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases

Cited by 24 publications

References 24 publications

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

Methods of Glioma Sample Processing for Molecular Diagnosis for the Glioma Tissue Bank Project

Opportunistic fungal pathogen Candida glabrata circulates between humans and yellow-legged gulls

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