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Digital PCR using micropatterned superporous absorbent array chips.

ABSTRACT: Digital PCR (dPCR) is an emerging technology for genetic analysis and clinical diagnostics. To facilitate the widespread application of dPCR, here we developed a new micropatterned superporous absorbent array chip (?SAAC) which consists of an array of microwells packed with highly porous agarose microbeads. The packed beads construct a hierarchically porous microgel which confers superior water adsorption capacity to enable spontaneous filling of PDMS microwells for fluid compartmentalization without the need of sophisticated microfluidic equipment and operation expertise. Using large ?-DNA as the model template, we validated the ?SAAC for stochastic partitioning and quantitative digital detection of DNA molecules. Furthermore, as a proof-of-concept, we conducted dPCR detection and single-molecule sequencing of a mutation prevalent in blood cancer, the chromosomal translocation t(14;18), demonstrating the feasibility of the ?SAAC for analysis of disease-associated mutations. These experiments were carried out using the standard molecular biology techniques and instruments. Because of its low cost, ease of fabrication, and equipment-free liquid partitioning, the ?SAAC is readily adaptable to general lab settings, which could significantly facilitate the widespread application of dPCR technology in basic research and clinical practice.

SUBMITTER: Wang Y

PROVIDER: S-EPMC4899130 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Digital PCR using micropatterned superporous absorbent array chips.

Wang Yazhen Y Southard Kristopher M KM Zeng Yong Y

The Analyst 20160324 12

Digital PCR (dPCR) is an emerging technology for genetic analysis and clinical diagnostics. To facilitate the widespread application of dPCR, here we developed a new micropatterned superporous absorbent array chip (μSAAC) which consists of an array of microwells packed with highly porous agarose microbeads. The packed beads construct a hierarchically porous microgel which confers superior water adsorption capacity to enable spontaneous filling of PDMS microwells for fluid compartmentalization wi ...[more]

PMID: 27010726

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Project description:The human rhinovirus (RV) is a positive-stranded RNA virus that causes respiratory tract diseases affecting both the upper and lower halves of the respiratory system. RV enhances its replication by concentrating RNA synthesis within a modified host membrane in an intracellular compartment. RV infections often occur alongside infections caused by other respiratory viruses, and the RV virus may remain asymptomatic for extended periods. Alongside qualitative detection, it is essential to accurately quantify RV RNA from clinical samples to explore the relationships between RV viral load, infections caused by the virus, and the resulting symptoms observed in patients. A reference material (RM) is required for quality evaluation, the performance evaluation of molecular diagnostic products, and evaluation of antiviral agents in the laboratory. The preparation process for the RM involves creating an RV RNA mixture by combining RV viral RNA with RNA storage solution and matrix. The resulting RV RNA mixture is scaled up to a volume of 25 mL, then dispensed at 100 µL per vial and stored at -80 °C. The process of measuring the stability and homogeneity of RV RMs was conducted by employing reverse transcription droplet digital polymerase chain reaction (RT-ddPCR). Digital PCR is useful for the analysis of standards and can help to improve measurement compatibility: it represents the equivalence of a series of outcomes for reference materials and samples being analyzed when a few measurement procedures are employed, enabling objective comparisons between quantitative findings obtained through various experiments. The number of copies value represents a measured result of approximately 1.6 × 105 copies/μL. The RM has about an 11% bottle-to-bottle homogeneity and shows stable results for 1 week at temperatures of 4 °C and -20 °C and for 12 months at a temperature of -80 °C. The developed RM can enhance the dependability of RV molecular tests by providing a precise reference value for the absolute copy number of a viral target gene. Additionally, it can serve as a reference for diverse studies.

Dataset Information

Digital PCR using micropatterned superporous absorbent array chips.

Publications

Digital PCR using micropatterned superporous absorbent array chips.

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