Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing

Olwagen, Courtney P.; Adrian, Peter V.; Madhi, Shabir А.

doi:10.1038/s41598-019-42846-y

Cited by 20 publications

(25 citation statements)

References 35 publications

(40 reference statements)

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“…Steady state mRNA levels from the selected gene loci were determined by RT-qPCR using the BIOMARK HD System (Fluidigm Corporations, South San Francisco, CA, USA) as previously described ( 30 ) at a cDNA concentration of 13.9 ng/μL. Negative controls and seven dilutions of the positive control sample described above were incorporated, each dilution being 3-fold and ranging from 0.17 to 125 ng/μL.…”

Section: Methodsmentioning

confidence: 99%

Cellular Proliferation of Equine Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells Decline With Increasing Donor Age

2020

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Background: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stem cells (MSCs) are used increasingly for autologous cell therapy in equine practice to treat musculoskeletal and other injuries. Current recommendations often call for 10–100 million MSCs per treatment, necessitating the expansion of primary cells in culture prior to therapeutic use. Of concern, human and rodent studies have shown a decline of both MSC recovery from sampled tissue and in vitro proliferative capacity with increasing donor age. This may be problematic for applications of autologous cell-based therapies in the important equine demographic of older patients.Objectives: To investigate the effect of donor age on the cellular proliferation of equine BM- and AT-MSCs.Study Design:In vitro study.Methods: BM- and AT-MSCs and dermal fibroblasts (biological control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15–17 months), adult (5–8 years), middle-aged (12–18 years), and geriatric (≥22 years). Proliferation of the cells was tested using an EdU incorporation assay and steady state mRNA levels measured for targeted proliferation, aging, and senescence biomarkers.Results: The cellular proliferation of equine BM- and AT-MSCs declined significantly in the geriatric cohort relative to the younger age groups. Proliferation levels in the two MSC types were equally affected by donor age. Analysis of steady state mRNA levels showed an up-regulation in tumor suppressors, apoptotic genes, and multiple growth factors in MSCs from old horses, and a down-regulation of some pro-cycling genes with a few differences between cell types.Main Limitations: Potential age-dependent differences in cell function parameters relevant to cell-therapy application were not investigated.Conclusions: The cellular proliferation of equine BM- and AT-MSCs declined at advanced donor ages. High levels of in vitro proliferation were observed in both MSC types from horses in the age groups below 18 years of age.

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Section: Methodsmentioning

confidence: 99%

Cellular Proliferation of Equine Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells Decline With Increasing Donor Age

2020

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“…The performance (sensitivity, speci city, and limit of detection) of the serogroup 6, 18, and 22 reaction-sets in the Fluidigm platform is comparable with other high-throughput strategies including the TaqMan Array Cards and the same Fluidigm platform including using different real-time PCR chemistry 10,32,33 . The performance of this reaction-set demonstrates that modi ed primers (DPO) or probes (LNA) are easily adapted to highthroughput real-time PCR, including where speci c target pre-ampli cation is undertaken in multiplex (up to 34 assay-sets per tube).…”

Section: Discussionmentioning

confidence: 86%

“…We included oligonucleotide primers and dye-labelled Minor Grove Binding (MGB) probes targeting serogroup 6 (6A/B/C/D), sub-group 6C/D, subgroup 18A/B/C, and serogroup 22 (22A/F) from published sequences 11,37 that have been optimized previously on the Biomark™ HD platform real-time PCR (Fluidigm) 32 in the reaction-set.…”

Section: Primer Designmentioning

confidence: 99%

“…The serogroup 6, serogroup 18 and serogroup 22 assay-sets designed in this study were included in a highthroughput nano uidic real-time PCR serotyping panel on the Fluidigm platform that was optimised as described previously 32 and here was expanded to include other published assay-sets to increase the number of individual serotypes detected. The Fluidigm work ow is summarized in Fig.…”

Section: Fluidigm Real-time Qpcrmentioning

confidence: 99%

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High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides.

Downs

Madhi

Merwe

et al. 2021

Preprint

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Current real-time Polymerase Chain Reaction (qPCR) methods are unable to distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009-11), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92–101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/ml of sample. There was high concordance (Kappa = 0.73–0.92); sensitivity (85–100%) and specificity (96–100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

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“…Probes to detect supplementary targets such as other RNA viruses, or host response genes can also be included. This flexibility presents a clear added value for both clinical monitoring of viral and bacterial pathogens as well as for research studies [ 6 , 7 ]. Another advantage of this system resides in the low amount of reagents needed compared to classical real-time PCR machines.…”

Section: Introductionmentioning

confidence: 99%

Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

et al. 2021

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The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

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Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing

Cited by 20 publications

References 35 publications

Cellular Proliferation of Equine Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells Decline With Increasing Donor Age

Cellular Proliferation of Equine Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells Decline With Increasing Donor Age

High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides.

Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

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