A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies

Tan, Chianru; Chen, Xihua; Wang, Fang; Wang, Dong; Cao, Zongfu; Zhu, Xiurui; Chen, Lu; Yang, Wei; Gao, Na; Gao, Huafang; Guo, Yong; Zhu, Lingxiang

doi:10.1039/c8an02018c

Cited by 44 publications

(53 citation statements)

References 34 publications

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“…The droplet digital polymerase chain reaction (ddPCR) is a novel molecular technique that has been developed to improve the sensitivity and quantification of rare target DNA sequences, for example in liquid biopsies for cancer monitoring, non‐invasive prenatal testing for genetic abnormalities and detection of DNA contaminants in bioprocessing (Hussain et al , ; Oellerich et al , ; Postel et al , ; Zhang et al , ; Wang et al , ; Tan et al , ; Galimberti et al , ). After DNA isolation, ddPCR generates approximately 20 000 miniscule droplets, which enriches target DNA sequences by reducing the competition with high‐copy templates (Fig.…”

Section: Introductionmentioning

confidence: 99%

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

Wouters

Dalloyaux

Christenhusz

et al. 2019

Microbial Biotechnology

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Summary The droplet digital polymerase chain reaction (ddPCR) is a novel molecular technique that allows rapid quantification of rare target DNA sequences. Aim of this study was to explore the feasibility of the ddPCR technique to detect pathogen DNA in whole blood and to assess the diagnostic accuracy of ddPCR to detect bloodstream infections (BSIs), benchmarked against blood cultures. Broad‐range primers and probes were designed to detect bacterial 16S rRNA (and Gram stain for differentiation) and fungal 28S rRNA. To determine the detection limit of ddPCR, 10‐fold serial dilutions of E. coli and C. albicans were spiked in both PBS and whole blood. The diagnostic accuracy of ddPCR was tested in historically collected frozen blood samples from adult patients suspected of a BSI and compared with blood cultures. Analyses were independently performed by two research analysts. Outcomes included sensitivity and specificity of ddPCR. Within 4 h, blood samples were drawn, and DNA was isolated and analysed. The ddPCR detection limit was approximately 1–2 bacteria or fungi per ddPCR reaction. In total, 45 blood samples were collected from patients, of which 15 (33%) presented with positive blood cultures. The overall sensitivity of ddPCR was 80% (95% CI 52–96) and specificity 87% (95% CI 69–96). In conclusion, the ddPCR technique has considerable potential and is able to detect very low amounts of pathogen DNA in whole blood within 4 h. Currently, ddPCR has a reasonable sensitivity and specificity, but requires further optimization to make it more useful for clinical practice.

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Section: Introductionmentioning

confidence: 99%

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

Wouters

Dalloyaux

Christenhusz

et al. 2019

Microbial Biotechnology

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“…Amplicon-based methods using sequence-specific primers have been proposed as an alternative to WGS for the assessment of aneuploidy 11-13 . Amplicon-based protocols offer many advantages over WGS (or exome sequencing), including a simpler workflow that does not require library construction, a reduced requirement for input DNA, and a simplified computational analysis.…”

mentioning

confidence: 99%

Assessing aneuploidy with repetitive element sequencing

Douville

Cohen

Ptak

et al. 2019

Preprint

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We report a sensitive PCR-based assay that can detect aneuploidy in samples containing as little as 3 picograms of DNA. Using a single primer pair, we amplified ∼750,000 amplicons distributed throughout the genome Aneuploidy was detected in 49% of liquid biopsies from a total of 883 non-metastatic cancers of eight different types. Combining aneuploidy with somatic mutation detection and eight standard protein biomarkers yielded a median sensitivity of 80% at 99% specificity.

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“…Using this system, researchers identified 39 differently transcribed cell populations by analyzing the transcriptome of 44 808 mouse retinal cells, and created a molecular map of gene regulatory linkages for known retinal cell types and new cell subtypes (Figure b) . In addition, researchers took advantage of droplet digital PCR (ddPCR) and developed several noninvasive methods for the screening of hereditary hearing loss‐related mutations and detection of fetal aneuploidies (an abnormal number of chromosomes) . Nevertheless, the workflow of this kind of method is relatively complicate, hindering its wider application in some specific situation (e.g., rare allele detection and DNA methylation analysis) .…”

Section: Microfluidics‐based Bioanalysismentioning

confidence: 99%

Microfluidics for Biomedical Analysis

et al. 2019

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Many new techniques and materials have been applied to microfluidic systems in the past decade, and integrated microfluidic chips have become powerful platforms for realizing highly sensitive, high throughput, and low‐cost analysis. This article reviews the latest advancements of microfluidic platforms for biological and biomedical analysis in the fields of fundamental biology research, clinical application, food safety, and environmental monitoring, with a focus on nucleic acid‐based molecular diagnosis and protein‐based immunodiagnosis. The combination of microfluidics with advanced techniques (e.g., nanotechnology, 3D printing, deep learning) would offer innovative new tools for bioanalyses. The current challenges and the future developments of microfluidic technologies for clinical applications are also discussed. It is anticipated that this review will be beneficial to promoting microfluidics toward a broad range of applications in the future.

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A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies

Abstract: Using universal locked nucleic acid probes, a high multiplexing ddPCR-based NIPT was developed to reliably identify fetal aneuploidies.

Cited by 44 publications

References 34 publications

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

Assessing aneuploidy with repetitive element sequencing

Microfluidics for Biomedical Analysis

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