Application Notes: App Note

Multiplexed Digital PCR for Non-Invasive Prenatal Trisomy Screening

Background

Aneuploidy is a genetic condition in which a person has missing or extra copies of chromosomes. The most common fetal aneuploidies are trisomies in which one chromosome has an additional copy. Of these, Patau syndrome (trisomy 13), Edwards syndrome (trisomy 18) and Down syndrome (trisomy 21) are the 3 most common types. While the discovery of fetal cell-free DNA (cfDNA) has enabled early detection of trisomies by looking for discrepancies in the mother’s blood, high costs have limited the availability of trisomy screening.

With recent improvements, digital PCR has the potential to become the standard of care for trisomy NIPT by providing accurate quantification, fast turnaround time and lower cost. In this study, we demonstrate the performance of a novel 4-color multiplexing NIPT trisomy test on the Absolute Q dPCR platform to simultaneously detect T13, T18 and T21.

Workflow features enable higher accuracy and sensitivity:

  • High partitioning consistency and low dead volume maximizes sample utilization
  • Digital pooling enables larger volumes of cfDNA to be analyzed across more partitions
  • 4 colors enables multi-target screening in a single reaction
Non-Invasive Prenatal Trisomy Screening
Figure 1. A simple workflow for the 4-color Atila NIPT assay testing on Combinati Absolute Q dPCR System with integrated digital PCR and data analysis.

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Emulsion-free Digital PCR Measurement of Wastewater Related Targets using the SARS-CoV-2 Wastewater Surveillance 4-plex Assay

Background

Wastewater based epidemiology (WBE) enables tracking of biomarkers for specific pathogens to monitor for disease outbreak and spread. WBE’s utility in disease surveillance has been proven to be effective in monitoring for rare cases of disease.(1) To effectively monitor and quantity of SARS-CoV-2 viral targets from wastewater samples, it is critical to maximize the amount of information per testing reaction, minimize reagent waste, and control for external factors such as population size and sample processing efficiency.

Measuring wastewater related targets alongside robust controls can provide tangible metrics for normalization of results and help limit the impact of sample preparation variability. The SARS-CoV-2 Wastewater Surveillance 4-plex Assay was designed to detect the N1 and N2 SARS-CoV-2 viral RNA targets alongside the human fecal control, pepper mild mottle virus (PMMoV), and a spike-in sample process control, bovine coronavirus (BCoV).

In this study, we demonstrate SARS-CoV-2 detection and quantification alongside the normalization controls for four wastewater samples collected by the University of Arizona WEST center during their wastewater epidemiology testing efforts.

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  1. Asghar H, et al., Environmental surveillance for polioviruses in the global polio eradication initiative. J Infect Dis. 2014;210:S294–S303.

Testing Wastewater Samples for SARS-CoV-2 Using the Absolute Q

Background

As the COVID-19 pandemic continues to have a lasting global impact, effective methods for monitoring communities for early signs of disease spread are a critical need. Screening wastewater, or sewage, for the presence of SARS-CoV-2 viral RNA can be an effective orthogonal monitoring method in addition to ongoing clinical testing. Wastewater serves as pooled samples from members of a community and enables broad collection of surveillance data – even in areas that have limited access to healthcare or testing facilities. Because natural sewage is highly heterogeneous, a method capable of identifying very rare target RNA from a mixture of non-target nucleic acid molecules is required. While quantitative PCR (qPCR) is the current standard for COVID-19 clinical testing, the resulting data can be highly variable due to inadequate sample dilution or chemical contamination. These challenges have a significant impact on measurements of targets that are of low abundance.

In contrast, digital PCR (dPCR) is aptly suited for detecting SARS-CoV-2 targets from wastewater samples. Using dPCR, scientists divide the sample and assay mixture into a large number of independent small reactions, such that zero or one target molecule is present in any individual reaction. Digital PCR overcomes the problem of variability, reduces the impact of many PCR inhibitors, and eliminates the need for standard curves, thus improving accuracy and confidence in rare target detection.1 Digital PCR has been proven to be a more sensitive method of SARS-CoV-2 detection – producing fewer false negatives and demonstrating better performance for low viral load specimens.2

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  1. Sean C. Taylor et al. Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Scientific Reports. 2017 May 25;7(1):2409.
  2. Dong, Lianhua, et al. “Highly Accurate and Sensitive Diagnostic Detection of SARS-CoV-2 by Digital PCR.” MedRxiv, Cold Spring Harbor Laboratory Press, 1 Jan. 2020.

Using Digital PCR for Optimization of SARS-CoV-2 RNA Extraction Protocol

Background

RNA extraction is a critical step in COVID-19 molecular testing. Loss of viral RNA during the extraction step can result in false negatives. Therefore, optimization of the RNA extraction protocol to ensure consistent and high yield recovery of viral RNA could potentially improve COVID-19 testing accuracy. The goal of this study is to demonstrate how digital PCR can be used to optimize conditions for viral RNA extraction using verified molecular controls. Digital PCR may also be used as a quality control tool to monitor sample preparation consistency across facilities and labs.

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4-Color Discrimination of Multi-Allele Single Nucleotide Polymorphisms on the Absolute Q

Background

Single nucleotide variants (SNVs) or single nucleotide polymorphisms (SNPs), have been implicated in many diseases. The detection, quantification, and discrimination of SNPs has a myriad of relevant applications in precision medicine. Furthermore, multiplexing a reaction to identify more than one allele target per reaction maximizes the amount of data obtained from a single sample. With high specificity and absolute quantification capabilities, digital PCR (dPCR) offers a technical advantage over many SNP detection or genotyping methods.

The Absolute Q is a fully integrated 4-color digital PCR platform that automates all steps of a typical dPCR reaction including partitioning, thermal cycling, and data acquisition. The microfluidic array partitioning (MAP) plate provides routine and consistent generation of 20,000 identically sized partitions, dispersing over 95 percent of sample across each dPCR reaction, every time. Unlike many available digital PCR systems, the workflow is identical to qPCR, and generates digital PCR results in as little as 90 minutes.

To demonstrate 4-color optical multiplexing for single nucleotide difference discrimination, a 4-plex assay was designed in collaboration with Integrated DNA Technologies for a set of alleles in the CYP2C19 gene (rs12248560). The cytochrome P450 enzyme mediates the primary metabolism of many drugs. Polymorphisms in this gene alter metabolism of certain drug compounds. The polymorphism rs12248560, an ultra-fast metabolism phenotype, has been linked to more favorable outcomes for breast cancer patients receiving the drug tamoxifen.1

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1. Schroth W; Antoniadou L; Fritz P; Schwab M; Muerdter T; Zanger UM; Simon W; Eichelbaum M; Brauch H; “Breast Cancer Treatment Outcome with Adjuvant Tamoxifen Relative to Patient CYP2D6 and CYP2C19 Genotypes.” Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology, U.S. National Library of Medicine, pubmed.ncbi.nlm.nih.gov/18024866/.

Evaluation of CRISPR-Cas9 Mediated Genome Edits with the Absolute Q Digital PCR platform

Background

Genome editing through the use of the CRISPR/Cas9 system has become a tool which is widely used across many scientific disciplines. As more labs employ the use of CRISPR/Cas 9 to introduce customized changes to targets, it is equally critical to have a method of monitoring the success and efficiency of these gene editing processes.

Digital PCR, which provides higher precision quantification of nucleic acids, is aptly suited for the analysis of genome editing applications such as CRISPR/Cas9 mediated knock-ins and knock-outs. This is largely enabled by dPCR’s fundamental principle of absolute quantification, which provides quantification of nucleic acid targets without the need for orthogonal standard curves. This method of quantification is more consistent and more accurate, particularly for rare or low concentration targets. This application note highlights the Absolute Q digital PCR platform paired with a 2-probe assay design designed in collaboration with Integrated DNA Technologies (IDT) which was used to detect and quantify both CRISPR meditated knock in and knock outs with precision and accuracy. 

The Absolute Q is a vertically integrated digital PCR platform which enables a simplified digital PCR workflow – using a single instrument and a single consumable to deliver complete results in under 2 hours. The microfluidic array partitioning (MAP) plate provides routine and consistent generation of 20,000 identically sized partitions, dispersing over 95 percent of the sample across each dPCR reaction every time. Unlike many available digital PCR systems, the workflow is identical to qPCR. This means every user can generate consistent and accurate digital PCR every time with minimal hands-on steps. 

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Testing qPCR positive, negative and inconclusive COVID-19 clinical samples using digital PCR

Background

In response to the global outbreak of coronavirus disease 2019 (COVID-19) there is a high demand for sensitive, accurate and consistent tests. Although RT-qPCR has served as the standard of care diagnostic test for the detection of SARS-CoV-2 infection, RT-dPCR (reverse transcription digital PCR) has recently been shown to outperform the traditional method in terms of sensitivity and accuracy.(1,2)

False negative and questionable negative rates resulting in inconclusive SARS-CoV-2 results using the current screening methodologies (RT-qPCR) have varied over the course of the pandemic and have been reported to be as high as 20%.(3,4) Because of this, asymptomatic patients are at an elevated risk of unknowingly spreading the disease. In addition to the need for more sensitive screening methods, a technology enabling higher accuracy will be critical for screening in determining more accurate rates of re-infection.

A highly sensitive, orthogonal test method to help resolve inconclusive SARS-CoV-2 results will increase overall testing accuracy and may also help reduce community transmission. The Combinati Absolute Q with its industry leading accuracy is ideally suited for the disambiguation of questionable negative test outcomes.

The goal of this study was to compare the results obtained using the CDC RT-qPCR assay with a dPCR test on a series of clinical samples. In collaboration with USC Clinical Laboratories, Molecular Pathology at University of Southern California, nucleic acids extracted  from 19 clinical specimens from individuals who tested negative or were diagnosed with COVID-19 were tested on the Combinati Absolute Q dPCR Platform using the |Q|™ SARS-CoV-2 Triplex Assay.

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  1. Suo T, Liu X, Guo M, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv. March 2020:2020.02.29.20029439. doi:10.1101/2020.02.29.20029439
  2. Dong L, Zhou J, Niu C, et al. Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR. medRxiv. March 2020:2020.03.14.20036129. doi:10.1101/2020.03.14.20036129
  3. Woloshin, Steven, et al. “False Negative Tests for SARS-CoV-2 Infection – Challenges and Implications: NEJM.” New England Journal of Medicine, 22 May 2020, www.nejm.org/doi/full/10.1056/NEJMp2015897.
  4. Yu F, et al., “Quantitative Detection and Viral Load Analysis of SARS-CoV-2in Infected Patients”, Clin Infect Dis, 2020

|Q| SARS-CoV-2 Triplex Assay: Multiplexed 1-step RT-dPCR for Accurate Viral Target Detection

Reverse Transcription Digital PCR (RT-dPCR) enables higher accuracy quantification 

The COVID-19 pandemic has drawn heightened concern, with over eleven million positive SARS-CoV-2 cases confirmed worldwide by July 2020.1 RT-qPCR currently serves as the clinical standard for the detection of SARS-CoV-2 viral RNA and subsequent diagnosis of COVID-19. However in a recent study, it was demonstrated that digital PCR (dPCR) provided better sensitivity for rare viral targets and in turn identifying patients who ultimately were diagnosed with COVID-19.Reverse transcription digital PCR (RT-dPCR) is a valuable technique which enables improved consistency and lower limits of detection compared to qPCR. Quantification of extremely rare SARS-CoV-2 viral RNA target material is also possible without the need for a comparative standard curve.

The Combinati |Q| SARS-CoV-2 Triplex Assay was designed as a single tube solution for SARS-CoV-2 identification and quantification with an integrated control assay for human gDNA. This assay paired with the true 1-step RT-dPCR workflow of the Absolute Q dPCR platform enables integration of sample digitization, reverse transcription, PCR and data collection into a single instrument and can be completed in approximately 80 minutes.

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  1. (WHO), World Health Organization. “Coronavirus Disease (COVID-19) – Situation Report 169.” Coronavirus Disease (COVID-2019) Situation Reports, 7 July 2020, 10:00 CEST, www.who.int/docs/default-source/coronaviruse/situation-reports/20200707-covid-19-sitrep-169.pdf?sfvrsn=c6c69c88_2.
  2. Dong L, Zhou J, Niu C, et al. Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR. medRxiv. March 2020:2020.03.14.20036129. doi:10.1101/2020.03.14.20036129

Rare Allele Detection and Quantification Using IDT rhAmp SNP Genotyping System

Introduction

Precise and sensitive detection of mutation bearing DNA molecules can be critical to drug selection in cancer treatment. For instance, EGFR is an important monitoring target in the treatment of Non-small Cell Lung Carcinoma (NSCLC). Specifically, the presence of EGFR p.T790M mutation indicates tumor resistance to treatment with tyrosine kinase inhibitors (TKIs).1

Integrated DNA Technologies’ rhAmp SNP Genotyping System utilizes RNase H2-depended PCT (rhPCR), a twoenzyme PCR chemistry, which enables highly precise interrogation of SNPs within challenging genomic regions.2 The Combinati Absolute Q digital PCR (dPCR) system utilizes micro-molded plastic picoliter partitions (Figure 1) instead of oil/water emulsions, thus enabling flexibility to accommodate the rhAmp chemistry. For the first time, the IDT rhAmp assay performance was demonstrated on a micro-chamber array based digital PCR platform.

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  1. Moran C. (2011). Importance of molecular features of nonsmall cell lung cancer for choice of treatment. The American
    journal of pathology, 178(5), 1940–1948. https://doi.org/10.1016/j.
    ajpath.2010.12.057
  2. rhAmp SNP Genotyping System. https://www.idtdna.com/pages/
    products/qpcr-and-pcr/genotyping/rhamp-snp-genotyping

Precise BCR-ABL1 Quantification for Chronic Myeloid Leukemia (CML) Monitoring

Abstract

A novel digital PCR (dPCR) platform combining off-the-shelf reagents, a micro-molded plastic microfluidic consumable with a fully integrated single dPCR instrument was developed to address the needs for routine clinical diagnostics. This new platform offers a simplified workflow that enables: rapid time-to-answer; low potential for cross contamination; minimal sample waste; all within a single integrated instrument. Here we showcase the capability of this fully integrated platform to detect and quantify non-small cell lung carcinoma (NSCLC) rare genetic mutants (EGFR T790M) with precision cell-free DNA (cfDNA) standards. Next, we validated the platform with an established chronic myeloid leukemia (CML) fusion gene (BCR-ABL1) assay down to 0.01% mutant allele frequency to highlight the platform’s utility for precision cancer monitoring. Thirdly, using a juvenile myelomonocytic leukemia (JMML) patient-specific assay we demonstrate the ability to precisely track an individual cancer patient’s response to therapy and show the patient’s achievement of complete molecular remission. These three applications highlight the flexibility and utility of this novel fully integrated dPCR platform that has the potential to transform personalized medicine for cancer recurrence monitoring.

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