Virtual-Partition Digital PCR for High-Precision Chromosomal Counting Applications.

Digital PCR (dPCR) is the gold-standard analytical platform for rapid high-precision quantification of genomic fragments. However, current dPCR assays are generally limited to monitoring 1-2 analytes per sample, thereby limiting the platform's ability to address some clinical applications that require the simultaneous monitoring of 20-50 analytes per sample. Here, we present virtual-partition dPCR (VPdPCR), a novel analysis methodology enabling the detection of 10 or more target regions per color channel using conventional dPCR hardware and workflow. Furthermore, VPdPCR enables dPCR instruments to overcome upper quantitation limits caused by partitioning error. While traditional dPCR analysis establishes a single threshold to separate negative and positive partitions, VPdPCR establishes multiple thresholds to identify the number of unique targets present in each positive droplet based on fluorescence intensity. Each physical partition is then divided into a series of virtual partitions, and the resulting increase in partition count substantially decreases partitioning error. We present both a theoretical analysis of the advantages of VPdPCR and an experimental demonstration in the form of a 20-plex assay for noninvasive fetal aneuploidy testing. This demonstration assay─tested on 432 samples contrived from sheared cell-line DNA at multiple input concentrations and simulated fractions of euploid or trisomy-21 "fetal" DNA─is analyzed using both traditional dPCR thresholding and VPdPCR. VPdPCR analysis significantly lowers the variance of the chromosomal ratio across replicates and increases the accuracy of trisomy identification when compared to traditional dPCR, yielding > 98% single-well sensitivity and specificity. VPdPCR has substantial promise for increasing the utility of dPCR in applications requiring ultrahigh-precision quantitation.

Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR.

The gold standard of molecular pathogen detection is the quantitative polymerase chain reaction (qPCR). Modern qPCR instruments are capable of detecting 4-6 analytes in a single sample: one per optical detection channel. However, many clinical applications require multiplexing beyond this traditional single-well capacity, including the task of simultaneously testing for SARS-CoV-2 and other respiratory pathogens. This can be addressed by dividing a sample across multiple wells, or using technologies such as genomic sequencing and spatial arrays, but at the expense of significantly higher cost and lower throughput compared with single-well qPCR. These trade-offs represent unacceptable compromises in high-throughput screening scenarios such as SARS-CoV-2 testing. We demonstrate a novel method of detecting up to 20 targets per well with standard qPCR instrumentation: high-definition PCR (HDPCR). HDPCR combines TaqMan chemistry and familiar workflows with robust encoding to enable far higher levels of multiplexing on a traditional qPCR system without an increase in cost or reduction in throughput. We utilize HDPCR with a custom 20-Plex assay, an 8-Plex assay using unmodified predesigned single-plex assays from Integrated DNA Technologies and a 9-Plex pathogen panel inclusive of SARS-CoV-2 and other common respiratory viruses. All three assays were successful when tested on a variety of samples, with overall sample accuracies of 98.8, 98.3, and 100%, respectively. The HDPCR technology enables the large install base of qPCR instrumentation to perform mid-density multiplex diagnostics without modification to instrumentation or workflow, meeting the urgent need for increased diagnostic yield at an affordable price without sacrificing assay performance.

Resonance sonomanometry for noninvasive, continuous monitoring of blood pressure.

Cardiovascular disease is the leading cause of death worldwide. Existing methods for continuous, noninvasive blood pressure (BP) monitoring suffer from poor accuracy, uncomfortable form factors, or a need for frequent calibration, limiting their adoption. We introduce a new framework for continuous BP measurement that is noninvasive and calibration-free called resonance sonomanometry. The method uses ultrasound imaging to measure both the arterial dimensions and artery wall resonances that are induced by acoustic stimulation, which offers a direct measure of BP by a fully determined physical model. The approach and model are validated in vitro using arterial mock-ups and then in multiple arteries in human subjects. This approach offers the promise of robust continuous BP measurements, providing significant benefits for early diagnosis and treatment of cardiovascular disease.

Supercolor coding methods for large-scale multiplexing of biochemical assays.

We present a novel method for the encoding and decoding of multiplexed biochemical assays. The method enables a theoretically unlimited number of independent targets to be detected and uniquely identified in any combination in the same sample. For example, the method offers easy access to 12-plex and larger PCR assays, as contrasted to the current 4-plex assays. This advancement would allow for large panels of tests to be run simultaneously in the same sample, saving reagents, time, consumables, and manual labor, while also avoiding the traditional loss of sensitivity due to sample aliquoting. Thus, the presented method is a major technological breakthrough with far-reaching impact on biotechnology, biomedical science, and clinical diagnostics. Herein, we present the mathematical theory behind the method as well as its experimental proof of principle using Taqman PCR on sequences specific to infectious diseases.

A rapid, multiplex digital PCR assay to detect gene variants and fusions in non-small cell lung cancer.

Digital PCR (dPCR) is emerging as an ideal platform for the detection and tracking of genomic variants in cancer due to its high sensitivity and simple workflow. The growing number of clinically actionable cancer biomarkers creates a need for fast, accessible methods that allow for dense information content and high accuracy. Here, we describe a proof-of-concept amplitude modulation-based multiplex dPCR assay capable of detecting 12 single-nucleotide and insertion/deletion (indel) variants in EGFR, KRAS, BRAF, and ERBB2, 14 gene fusions in ALK, RET, ROS1, and NTRK1, and MET exon 14 skipping present in non-small cell lung cancer (NSCLC). We also demonstrate the use of multi-spectral target-signal encoding to improve the specificity of variant detection by reducing background noise by up to an order of magnitude. The assay reported an overall 100% positive percent agreement (PPA) and 98.5% negative percent agreement (NPA) compared with a sequencing-based assay in a cohort of 62 human formalin-fixed paraffin-embedded (FFPE) samples. In addition, the dPCR assay rescued actionable information in 10 samples that failed to sequence, highlighting the utility of a multiplexed dPCR assay as a potential reflex solution for challenging NSCLC samples.

Microfluidic blood filtration device.

Rapid decentralized biomedical diagnostics have become increasingly necessary in a medical environment of growing costs and mounting demands on healthcare personnel and infrastructure. Such diagnostics require low-cost novel devices that can operate at bedside or in doctor offices using small amounts of sample that can be extracted and processed on the spot. Thus, point-of-care sample preparation is an important component of the necessary diagnostic paradigm shift. We therefore introduce a microfluidic device which produces plasma from whole blood. The device is inexpensive, reliable, easy to fabricate, and requires only 3.5 kPa pressure to operate. The device is fully compatible with microfluidic diagnostic chips. The output 23-gauge microtube of the former can be directly plugged into the input ports of the latter allowing immediate applicability in practice as a sample-prep pre-stage to a variety of emergent microfluidic diagnostic devices. In addition, the shown approach of filter encapsulation in elastomer has principle importance as it is compatible with and applicable to microfluidic sample-prep integration with analytical stages within the same elastomeric chip. This can eventually lead to finger-prick blood tests in point-of-care settings.

Significant Expansion of Real-Time PCR Multiplexing with Traditional Chemistries using Amplitude Modulation.

The real time polymerase chain reaction (rtPCR) is an essential method for detecting nucleic acids that has a wide range of clinical and research applications. Current multiplexed rtPCR is capable of detecting four to six nucleic acid targets in a single sample. However, advances in clinical medicine are driving the need to measure many more targets at once. We demonstrate a novel method which significantly increases the multiplexing capability of any existing rtPCR instrument without new hardware, software, or chemistry. The technique works by varying the relative TaqMan probe concentrations amongst targets that are measured in a single fluorometric channel. Our fluorescent amplitude modulation method generates a unique rtPCR signature for every combination of targets present in a reaction. We demonstrate this technique by measuring nine different targets across three color channels with TaqMan reporting probes, yielding a detection accuracy of 98.9% across all combinations of targets. In principle this method could be extended to measure 6 or more targets per color channel across any number of color channels without loss in specificity.