Application Notes: Absolute Quantification

Digital PCR Wastewater Surveillance: Detect SARS-CoV-2 alongside human fecal and process controls

Background/Significance

Wastewater surveillance of SARS-CoV-2 has been shown to be a useful predictor of potential outbreaks. However, for meaningful interpretation of SARS-Cov2 data, both quantification accuracy and data normalization are critical. Moreover, reverse transcription qPCR (RT-qPCR) – the most commonly used method – reports a threshold cycle that requires a standard curve to provide quantitative information. RT-qPCR’s reliance on standard curves means the accuracy of the measurements depends directly on the accuracy and reproducibility of the reference materials used. These factors combined make interpretation of data on a broad scale extremely challenging. 

Digital PCR (dPCR) provides absolute quantification, and when combined with multi-colored multiplexing, can incorporate controls in a single reaction to provide normalized results for multiple targets. These results enable more accurate comparisons between samples with varying upstream sample preparation methodologies and more robust longitudinal monitoring.

Benefits of the Absolute Q for SARS-CoV-2 Wastewater Monitoring

  • Quantification of three wastewater-specific genomic targets in a single dPCR reaction
  • Integration of Human Fecal and Process Controls allows normalization and recovery efficiency to be calculated without additional reactions
  • Single instrument qPCR-like workflow in under 2 hours

The Combinati 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). In addition to these three targets, the assay also integrates the process control Bovine Coronavirus (BCoV). This inactivated virus, which is not generally present in community sewer systems, is spiked into the initial raw sewage sample before downstream processing. Its similarity to human SARS-CoV-2 allows it to be used as a surrogate to monitor the overall efficiency of sample processing.

Assay Design

The Combinati SARS-CoV-2 Wastewater Surveillance 4-plex assay combines four sets of primers/probes into a single multiplexed assay. The assay is composed of two genetic targets N1 and N2 on the SARS-COV-2 N gene, a matrix recovery target (aka. process control) on Bovine coronavirus genome (BCoV), and a Human fecal normalization control target on Pepper Mild Mottle virus (PMMoV). N1 and N2 have been reported to be sensitive and specific for quantifying SARS-CoV-2 RNA in wastewater. Bovine coronavirus is an enveloped virus with a single stranded RNA genome similar to SARS-CoV-2, but not usually present in wastewater. The plant pathogen Pepper Mild Mottle Virus (PMMoV), an indicator of human fecal pollution, is widespread and abundant in wastewater from the United States.

N1, N2, BCoV and PMMoV targets were labeled with FAM, HEX, TAMRA, and TYE665, respectively (Figure 1). All primers were checked for target sequence specificity using NCBI Primer-BLAST1. Primers and probes were also evaluated for primer dimers and cross primer interactions using Multiple Primer Analyzer (Thermo Fisher Scientific, Waltham, MA).

Absolute Q Workflow and Experiment Materials

After preparing the dPCR mix, 9µL of the reaction mixture was loaded into the MAP16 plate followed by an overlay of 15µL of isolation buffer (Figure 1). The prepared MAP16 plate was then loaded on the Absolute Q. Figure 2 details the thermal cycling and reagent preparation protocols for RT-dPCR on the Absolute Q.

Figure 1. Workflow for the SARS-CoV-2 Wastewater Surveillance 4-plex assay

Figure 2 (right). Absolute Q digital PCR thermal parameters and reagent preparation table

Assay Performance: Sensitivity

Accurate quantification of SARS-CoV-2 RNA is critical when comparing results between locations or performing longitudinal surveillance. We prepared and quantified a serial dilution of the commercially available SARS-CoV-2 control material (Exact Diagnostics, SKU: COV019), which contains both the N1 and N2 targets to demonstrate the quantification accuracy of the assay. 

The serial dilutions consisted of three 4-fold dilutions to simulate a range of viral RNA concentrations. These RNA dilutions were spiked into a constant background of BCoV and PMMoV control materials at approximately 500 and 1000 copies per reaction respectively. Figure 3 summarizes the results of this N gene assay sensitivity dilution series.

Figure 3. Results from serial 4-fold dilutions of Exact Diagnostic SARS-CoV-2 control material into a background of BCoV and PMMoV control material at approximately 500 and 1000 copies per reaction respectively. A) The X-axis represents the targeted copies/reaction and Y-axis represents the observed concentrations of the N gene targets, N1 (purple) and N2 (orange). Two-dimensional partition scatters for the N1 (FAM) and N2 (HEX) targets across the RNA control material dilution using 2µL each of the (B) stock control material, (C) 4X dilution (D) 16X dilution and (E) 64X dilution.

The targeted concentrations of the N1 and N2 genes across the dilution series were 550, 137.5, 34.4 and 8.5 copies per reaction with the observed concentrations reported in Table 1.  The concentrations are significantly correlated for both N1 and N2 with Pearson R2 values of 1.0 (p<0.001). Across each SARS-CoV-2 dilution point, both BCoV and PMMoV remained constant at 471.8 (±32.4) and 1194.5 (±77.3) copies per reaction respectively.

Table 1. Average observed N1 and N2 copies per reaction using the SARS-CoV-2 Wastewater Surveillance 4-plex assay. At least 3 replicates were run for each condition. In addition, a water control was included. In one of three NTCs, a single false positive partition was identified for N1 and N2.

Assay Performance – Specificity

To demonstrate the high specificity and low cross reactivity of the 4-plex assay, individual materials and mixtures of the target control materials were tested against the 4-plex assay. The 4-target PCR control material demonstrated successful amplification in all four targets (Figure 4a) and the no-template added negative control showed zero false positive amplification events (Figure 4b). Subsequent tests of individual target control materials demonstrated high specificity for the intended target for each assay component (Figure 4c-f).

Figure 4.  Partition amplification plots shown for each target N1 (blue channel, FAM), N2 (green channel, HEX), BCoV (yellow channel, TAMRA) and PMMoV (dark red channel, TYE665) by rows are: A) a PCR control 4-plex containing single stranded DNA (ssDNA) N1, N2, and PMMoV controls alongside inactivated BCoV RNA control material; B) water only no template control which yielded no false positives; C) N1 ssDNA control; D) N2 ssDNA control; E) PMMoV ssDNA control;  F) BCoV RNA control.

Using Human Fecal and Process Controls for Data Comparisons

Viral load present in wastewater can be impacted by a variety of factors, including differences in preparation methods as well as the total amount of human fecal matter present. Understanding the amount of human fecal matter relative to the quantitative measurement of SARS-CoV-2 enables more accurate data interpretation for community level testing. The SARS-CoV-2 Wastewater Surveillance 4-plex assay incorporates two orthogonal controls in order to help interpret results. Quantification of those controls (BCoV, process control and human fecal control PMMoV) in the same reaction as the SARS-CoV-2 N gene targets enables more precise comparison between samples. The following dataset illustrates one option for how the BCoV and PMMoC controls can be used to interpret SARS-CoV-2 wastewater-based epidemiology data.

Figure 5. Concentration of N2 (orange) and N1 (purple) SARS-CoV-2 targets in contrived samples. Using the SARS-CoV-2 Wastewater Surveillance 4-plex assay, three replicates were tested per contrived sample.

We tested four contrived samples using the SARS-CoV-2 Wastewater Surveillance 4-plex assay. All four samples demonstrated similar overall N1 and N2 quantities (Figure 5).  However, in the same four samples, the measured PMMoV concentration varies significantly (Figure 6a).

Figure 6. A) Initial concentration in copies per reaction of each contrived sample for the N1 (purple), N2 (orange) and PMMoV (red) targets. B) Chart reflects the concentration of N1 (purple) and N2 (orange) normalized to the median PMMoV value of 5357 copies of PMMoV.  Using the SARS-CoV-2 Wastewater Surveillance 4-plex assay, three replicates were tested per contrived sample.

In order to make viral load comparisons, normalization to the human fecal marker PMMoV can be used to account for information such as the size of the community sampled. In this example, the ratio of N1 or N2 quantities to the concentration of PMMoV was normalized to the median PMMoV concentration measured in this dataset (5357 PMMoV copies) as detailed in the Methods section. While Samples B and C demonstrated very similar levels of N1 and N2, their PMMoV concentrations varied substantially (Figure 6a). After normalization to the PMMoV median, Sample B had the highest relative abundance of SARS-CoV-2 targets (Figure 6b).  

Finally, the process control (BCoV) levels can be used to verify there are no large discrepancies in sample preparation efficiencies between the samples. As shown in Figure 7 the measured BCoV concentrations are comparable to one another across the four samples. Assuming an equivalent amount of control material was spiked into the native sample at the start of processing, this would indicate processing variations were minimal.

Figure 7. Concentration of BCoV across contrived samples using the SARS-CoV-2 Wastewater Surveillance 4-plex assay. Three replicates were tested per sample.

Summary

When comparing quantitative data, consistent measurement techniques that introduce as few variables as possible are essential. Digital PCR (dPCR), which provides absolute quantification of targets without standard curves, enables the quantification of all targets (including process and internal controls) to produce more accurate and more broadly comparable wastewater datasets – even when upstream preparation methods vary. 

The Combinati SARS-CoV-2 Wastewater Surveillance 4-plex assay was designed to detect and quantify SARS-CoV-2 viral targets while simultaneously providing normalization and recovery data in a single reaction. Using both human fecal markers and an orthogonal process control, sources of variability such as fecal load variation due to population levels or inconsistencies in sample processing can be accounted for. With high specificity, sensitivity, and best in class sample utilization of 95%, the Absolute Q provides more accurate and consistent quantification of these wastewater relevant targets.

Materials and Methods

Control Materials

Wet-lab validation of the assay has been performed using control materials. For assay specificity evaluation, single stranded DNA controls were used for the N1, N2 and PMMoV targets and Bovilis Coronavirus Calf Vaccine was used as the BCoV positive control. For assay sensitivity evaluation, the Exact Diagnostic SARS-CoV-2 RNA control material was used. For contrived samples, the N1, N2, BCoV, and PMMoV controls were mixed to create varying abundance ratios.

Normalization

To normalize the concentration of N1 and N2 with respect to PMMoV for the contrived sample experiment, the following steps were performed. First, the concentration of each target (N1, N2, and PMMoV) were multiplied by the reaction volume to calculate the total copies per reaction. Subsequently, the concentration of N1 or N2 was divided by the concentration of PMMoV to obtain a ratio. Finally, the ratio was multiplied by the median PMMoV concentration for the dataset.

References

  1. “Primer Designing Tool.” National Center for Biotechnology Information, U.S. National Library of Medicine, www.ncbi.nlm.nih.gov/tools/primer-blast/.

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.

Establishing the Limit of Detection for the |Q| SARS-CoV-2 Triplex Assay

Background

Widespread testing has been proven to be an important tactic to combat widespread infections during the COVID-19 pandemic. Many types of tests have been brought to the market in an effort to expand test availability to all corners of the globe. However, in order to choose the most appropriate option, it is important to understand and consider both the sensitivity and accuracy of the test in addition to its availability. False negatives could lead to an increase in community spread and significantly increase the risk of large scale outbreaks.

Limit of detection (LoD), also known as analytical sensitivity, is often used to describe the lowest concentration of input that can be reliably distinguished from a blank. In this study, we characterize the LoD of the Combinati |Q| SARS-CoV-2 RT-dPCR Triplex Kit by diluting reference materials into a pooled negative matrix to determine the lowest concentration at which the assay can reliably identify the sample as containing SARS-CoV-2 targets.

The traditional quantitative PCR (qPCR) approach, the current gold standard for COVID-19 diagnosis, generates results in terms of Ct or Cq. These values do not provide quantitative measurements of the virus without a standard curve. Instead, a predetermined qPCR threshold result determines if a sample is deemed positive or negative. In contrast to this binary result provided by qPCR, digital PCR (dPCR) provides absolute quantification of nucleic acid targets without a standard curve. Each dPCR assay provides a quantitative measure of the targets present in the original sample. In this study, we present quantitative dPCR data to demonstrate the ability to use the Combinati |Q| SARS-CoV-2 RT-dPCR Triplex Kit for applications that look to quantify viral load changes between samples or over time.

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Experimental Protocol

Serial dilution of input materials: Synthetic virus from SeraCare (AccuPlex™ SARS-CoV-2 Reference Material Kit, Cat No. 0505-0126) was serially diluted into a pooled negative swab matrix (VTM/UTM). The samples contained target concentrations from 1600cp/mL to 50cp/mL in 2-fold dilutions.

Nucleic acid purification: The Promega Maxwell RSC16 (Cat No. AS4500) and the Maxwell® RSC Viral Total Nucleic Acid Purification Kit (AS1330) were used to extract RNA from the dilution series samples. For each extraction, 150µL of sample input volume and 60µL elution volume were used.

Digital PCR protocol: For each dPCR run, 6.5µL of extracted sample was combined with 2.5µL of RT-dPCR MasterMix and 1µL of the Triplex Assay. 9µL of the reaction mixture was then loaded into a single well of the MAP16 consumable. Each MAP16 plate run included one NTC (no template control) to ensure that no contamination occurred during testing.

Thermal cycling protocol for the Absolute Q Digital PCR Platform is shown below in Table 1.

Table 1 Thermocycling Parameters

Interpretation of assay results: The determination of whether a sample is “positive” for SARS-CoV-2 was made according to Table 2.

Table 2 Interpretation of Assay Results

As described in the table, any sample that contained two or more positive partitions for either N1 or N2 target is considered positive for SARS-CoV-2.

Limit of detection determination and confirmation: Two sets of experiments were performed to determine the LoD. First, nine replicates for each of the dilutions from 1600cp/mL to 50cp/mL were tested to determine the preliminary LoD. Subsequently, the three lowest concentrations that demonstrated positive signal for all nine replicates (100% accuracy) were selected for additional testing. For each of these concentrations, 20 extraction replicates were tested to confirm the limit of detection.

Results

Preliminary LoD determination: Results from the LoD determination experiment are summarized below in Table 3.

Table 3 Preliminary LoD Determination Results

For all concentrations down to 200cp/mL, nine out of nine replicates (100%) resulted in positive calls for both N1 and N2 targets. For 100cp/mL input, only four out nine replicates were called correctly for N1 and six out of nine replicates were called correctly for N2. Based on these results, the three concentrations selected for the confirmation experiment were 800cp/mL, 400cp/mL, and 200cp/mL.

LoD confirmation: For LoD confirmation, 20 extraction replicates were performed for each of the three concentrations selected. The LoD is defined as the lowest input concentration that results in greater than or equal to 95% of all true positive replicates testing positive for SARS-CoV-2. Results for the confirmation experiment are summarized below in Table 4.

Table 4 Confirmation of LoD

The LoD was determined to be 200 cp/mL, as 20 out of 20 replicates were correctly identified as positive for SARS-CoV-2 for both N1 and N2.

Quantitative measurement of serially diluted samples: As dPCR provides absolute measurements instead of a cycle number, small changes can be accurately detected and quantified. Figure 1 shows the number of positive partitions for each of the dilutions used in the preliminary LOD study.

Figure 1. Number of Positive Partitions with Various Input Concentrations

A linear relationship between the input concentration and number of positive partitions detected was identified (N1 R2 = 0.994, N2 R2 = 0.993). This provides strong evidence for the feasibility of accurate and precise quantitative monitoring of viral presence changes using the Absolute Q Digital PCR Platform.

Results

In this study, we established the limit of detection of the Combinati |Q| SARS-CoV-2 Triplex Kit as 200 cp/mL and defined the protocol used to determine the LoD. Additionally, we demonstrated the ability to accurately quantify across a large range of input sample. In summary, the Combinati |Q| SARS-CoV-2 RT-dPCR Triplex kit combined with the Absolute Q Digital PCR Platform enabled highly sensitive detection and quantification of SARS-CoV-2 when coupled with the Promega RSC for nucleic acid purification. The quantitative measurement provided by the assay can be used for a wide range of applications, including tracking viral load changes and wastewater monitoring.

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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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 processes.

Digital PCR 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. Using two separate assays, this application note highlights how the Absolute Q digital PCR platform can be used to study the efficiency of CRISPR mediated genome edits.

The Absolute Q is the only vertically integrated, single instrument, digital PCR platform. 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, generating digital PCR results in as little as 90 minutes.

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Combinati |Q| SARS-CoV-2 Triplex Assay Digital PCR Protocol

Introduction

The rapid outbreak of COVID-19 originating in Wuhan, China has mobilized unprecedented response to the pandemic across the globe. Numerous diagnostic tests have been deployed to aid in control of disease spread. Positive control materials are required for assay development and to assess overall consistency. To ensure that COVID-19 tests have consistent limits of detection, accurate quantitative measurement of these control materials is critical.

Highlights

  • The Absolute Q provides best-in-class nucleic acid analysis with a complete 90 minute walk-away dPCR workflow.
  • Linear dynamic range verified down to 10 SARS-CoV-2 copies per reaction volume
  • Absolute quantification of reference material will ensure consistent assay performance and disease reporting
  • Accurate dPCR quantification of SARS-CoV-2 targets will benefit the global pandemic response.

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1-Step Reverse Transcription Digital PCR (RT-dPCR) in Under 2 Hours

Background

Reverse Transcription PCR (RT-PCR) is an important tool that allows the assessment of nucleic acid targets that are present in the form of RNA. It has a wide range of applications including gene expression and detection of RNA viruses. During 1-step RT-PCR, reverse transcription of RNA to cDNA occurs in the same reaction vessel as the PCR, which is especially important for clinical applications as the reduced manual handling improves consistency and reduces time to result. Reverse transcription digital PCR (dPCR) further improves the technique by making quantification of extremely rare target material possible without the need for a comparative standard curve – thus enabling better overall consistency and lower limits of detection. Furthermore, recent data suggests dPCR outperforms qPCR in the detection of viral targets such as the widespread SARS-CoV-2 virus.1

The Combinati Absolute Q is a novel 4-color dPCR platform with a complete workflow identical to qPCR. This system overcomes many challenges presented by current dPCR workflows. For example, dPCR typically requires a minimum of 2 instruments to execute the thermal cycling and data collection steps separately. This increases both the time to answer and hands on time as the user is required to move the samples from one stopping point to the next. The Absolute Q’s unique architecture allows it to handle reagent partitioning, reverse transcription, thermal cycling and data collection all on a single instrument and single consumable, enabling a true 1-step RT-dPCR workflow in under 2 hours. In this technical note, we showcase 1-step RT-dPCR on the Absolute Q using the |Q| SARS-CoV-2 Triplex Assay using an RNA-based reference material.

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Workflow and Materials

The SARS-CoV-2 Triplex Assay was designed using published CDC sequences as a single tube solution for SARS-CoV-2 identification and quantification with an integrated control assay for human gDNA. Using the Exact Diagnostics SARS-CoV-2 control material as input, we prepared the SARS-CoV-2 Triplex Assay according to table 1. In addition to quantification of the RNA-based standard as a proof of concept for 1-step RT-dPCR, human genomic DNA alone and water controls were included.

Table 1. 1-step RT-PCR Reaction mix formula.

*1µL of the control standard was loaded per reaction directly from stock. For negative control using human genomic DNA, 50 nanograms were loaded per reaction. Water was adjusted to accommodate the changes in sample volume.

Absolute Q Workflow

After preparing the dPCR mix, 10µL of the reaction mixture was loaded into the MAP16 plate followed by an overlay of 10µL of isolation buffer. The prepared MAP16 plate was then loaded on the Absolute Q. Table 2 details the thermal cycling protocol for RT-dPCR on the Absolute Q.

Table 2. 1-step RT-PCR parameters on the Absolute Q

Table 2. 1-step RT-PCR parameters on the Absolute Q

Quantification of SARS-CoV-2 Targets
Using Reference Materials

Unlike traditional RT-qPCR, RT-dPCR does not require a standard curve or reference sample to identify and quantify targets. The SARS-CoV-2 standard contains synthetic RNA targets from 5 genes of the novel coronavirus and human genomic DNA, which can be used to validate extraction methods. The Combinati SARS-CoV-2 Triplex assay targets the N1 and N2 gene sequences as well as the human RnaseP gene. As a demonstration of the consistency of one-step RT-dPCR using the control RNA as input, we performed the viral quantitation assay in duplicate across 4 separate instruments for a total of 8 replicates using this material.

We identified all three targets in the sample across 8 replicates and saw very few false positives within negative control reactions. We calculated the average concentration of viral and human targets and the associated standard deviation of the SARS-CoV-2 standard in copies per microliter – N1: 358.1 cp/µL (± 22.5), N2: 333.9 cp/µL (± 17.5) and RnaseP: 323.1 cp/µL (± 18.7). Fewer than one positive partition per dPCR reaction was identified on average across all replicate no template control reactions. For the human male control reactions, the values were: N1: 0.0 cp/µL (± 0.0), N2: 0.3 cp/µL (± 0.8). For water only, no template control reactions, the values were: N1: 0.4 cp/µL (± 0.9), N2: 0.1 cp/µL (± 0.4), RnaseP: 0.0 cp/µL (01770.0). Quantitation results were consistent across all four instruments (Figure 1).

Figure 1. Cross instrument Absolute Q quantification consistency using 1-step RT-dPCR. Data shown are the results of the Combinati SARS-CoV-2 Triplex Probe Assay testing the RNA-based SARS-CoV-2 Standard Control ((Exact Diagnostics) and 50 nanograms of human male control DNA (Promega) as a negative control for viral targets. Reactions were run in duplicate for each control material across four instruments for a total of eight replicates each.

Figure 1. Cross instrument Absolute Q quantification consistency using 1-step RT-dPCR. Data shown are the results of the Combinati SARS-CoV-2 Triplex Probe Assay testing the RNA-based SARS-CoV-2 Standard Control ((Exact Diagnostics) and 50 nanograms of human male control DNA (Promega) as a negative control for viral targets. Reactions were run in duplicate for each control material across four instruments for a total of eight replicates each.

Summary

The Absolute Q dPCR platform and its 1-step RT-dPCR technology have broad implications for characterizing infectious diseases beyond COVID-19. The versatile platform can be adapted to a wide range of nucleic acid detection applications requiring absolute quantification. The Absolute Q simplifies dPCR with best-in-class data consistency, a short sample-to-answer time, and flexible multi-color multiplexing capabilities. Combinati aims to lower the barrier to bring dPCR into the lab to accelerate the response to global public health emergencies such as the COVID-19 pandemic.

References

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

MAP16 dPCR Plate Flexibility: Iterative Assay Optimization Using a Single Plate

Highlights

  • Experiment flexibility to use a single consumable up to four times
  • Optimization of time allowed for annealing/extension step for a duplex BCR-ABL1 Assay
  • Maintained partition consistencies for iterative use

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The MAP16 consumable was used four times, using one column per experiment to optimize the annealing/extension time for a FAM/HEX multiplexed assay.

Figure 1. The MAP16 consumable was used four times, using one column per experiment to optimize the annealing/extension time for a FAM/HEX multiplexed assay.

Combinati’s patented Microfluidic Array Partitioning (MAP) technology utilizes fixed microchamber arrays and positive pneumatic pressure to partition reagents and perform digital PCR, instead of using fluid-shearing to generate droplets. Each MAP16 plate consists of a 4-unit by 4-unit grid of dPCR reaction units – each unit containing 20,480 fixed partitions. The plate was designed to enable flexibility – meaning up to 16 samples may be used simultaneously or as few as four units, i.e. one column, can be loaded and run at a time without sacrificing data quality. This flexibility can be useful for applications in which lower throughput for dPCR is desired or iterative assay optimization, demonstrated here, is required.

Method Details

In this tech note, an iterative test was performed on a single MAP16 plate to optimize the time allowed for the extension step of PCR for a BCR-ABL1 assay, which detects a gene fusion present in 95% of chronic myeloid leukemia patients. To showcase experiment flexibility of the consumable for repeat uses, we ran four sequential dPCR runs, modifying the extension step of PCR to be 0, 15, 30 and 45 seconds. We compared the final calculated concentration of target as well as compared the fluorescent intensity across each condition. To evaluate the integrity of the MAP plate across successive runs, we calculated the total number of partitions analyzed per condition.

We selected the BCR-ABL pDNA calibrant (Sigma, Cat:ERMAD623), a plasmid containing target sequences for both BCR-ABL1 and ABL1. ERM(R) certification of this well-characterized reference material ensures reliability and comparability of the results. We used a published duplex assay targeting the BCR-ABL1 (FAM) and ABL-1 (HEX) sequences respectively1, and prepared the assay using the Combinati 2X MasterMix. Each reaction contained a final target of 500 copies/µL. Using one column at a time, four replicates were run, and the concentration of each target was quantified in copies/µL. The reagent mix recipe and the dPCR protocol are described in Table 1.

Table 1. dPCR reagent preparation

Table 1. dPCR reagent preparation

For each partition, both low reagent volume and close proximity to the heated surface contribute to PCR robustness at a variety of extension times. Typically, the suggestion for extension time is approximately one minute per 1000 bases. In this study, we test the performance of the duplex assay at increasing extension time intervals (Table 2). In each of the four successive runs, a different column was utilized to evaluate the effects of changing the time allowed for annealing/extension step, starting at 0 seconds and increasing by 15 seconds with each run (Figure 1).

Table 2. Absolute Q dPCR thermal protocol

Table 2. Absolute Q dPCR thermal protocol

Results

The quantification results for both the BCR-ABL1 (FAM) and ABL1 (HEX) targets across the four different extension times (0 seconds, 15 seconds, 30 seconds, and 45 seconds) are shown in Figure 2, together with the representative 2D scatter plots. Extension times at 15 seconds or longer produced accurate quantification, while extension times of 30 seconds or greater provided the best separation between positive and negative partition clusters.

Figure 2. (A) Concentration of multiplex assay targets in the FAM and HEX channels. Colored bars indicate the various extension time used for each condition. Error bars represent the standard deviation, and mean values are noted at the top of each bar.

Figure 2A. Concentration of multiplex assay targets in the FAM and HEX channels. Colored bars indicate the various extension time used for each condition. Error bars represent the standard deviation, and mean values are noted at the top of each bar.

Figure 2B. Two-dimensional dPCR scatter plot data from a single representative reaction per condition. Extension time used denoted at the top.

Figure 2B. Two-dimensional dPCR scatter plot data from a single representative reaction per condition. Extension time used denoted at the top.

The industry standard “targeted minimum” number of analyzed dPCR partitions is typically 20,000. In addition to consistent quantification across repeated use of the same MAP16 plate, the average total number of partitions analyzed per unit remains well above the targeted minimum at 20,252 (±165) partitions per reaction. Figure 3 denotes the average number of accepted partitions and associated standard deviation for the entire plate used, as well as the average per run. Since each dPCR run for this assay requires 40 cycles of PCR, after the fourth run, the partitions in the last column have been exposed to thermal changes for an aggregate of 160 cycles. Even so, the MAP plate yields consistent numbers of acceptable partitions well above 20,000 per unit even in later runs (Figure 3).

Figure 3. Total partitions accepted for analysis by Combinati |Q| Analysis software for one MAP16 plate run 4 separate times to test the effect of extension time on dPCR assay performance. Results from all 4 runs are shown in the first column, and the results of individual runs are shown in subsequent columns. Each point represents the total partition yield from one dPCR unit.

Figure 3. Total partitions accepted for analysis by Combinati |Q| Analysis software for one MAP16 plate run 4 separate times to test the effect of extension time on dPCR assay performance. Results from all 4 runs are shown in the first column, and the results of individual runs are shown in subsequent columns. Each point represents the total partition yield from one dPCR unit.

Summary

Microfluidic Array Partitioning (MAP) technology enhances dPCR. With a simple workflow and highly consistent performance, the MAP plate enables flexibility in experimental design and optimization of dPCR assay conditions without sacrificing robustness.

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