Application Notes: High Multiplexity

Using in-line process controls to evaluate sample processing workflow efficiency

Introduction

Environmental monitoring of wastewater samples for the presence of SARS-CoV-2 has demonstrated the potential to provide population level estimates of COVID-19 disease burden. 1 While routine monitoring of community wastewater samples for the presence of SARS-CoV-2 viral targets can be complimentary to clinical testing of individuals, quantifying dynamic changes in the number of viral RNA molecules can help identify trends in potential COVID-19 cases within the community. It’s important to recognize the variability arising from differences in sample preparation methods to better track the changes in viral target concentration over time. Process controls are widely used to address sources of variability. Spike in controls, such as Bovine Coronavirus, is one of the most commonly employed targets to provide insight into this variability in wastewater surveillance efforts.

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Digital PCR provides more accurate quantitative results

Since dPCR provides standard curve-free absolute quantification of targets, the resulting quantitative data is more accurate and more reproducible than typical qPCR methods. Multiplexing provides additional quantitative data that can be leveraged for normalization of downstream results and help limit the impact of upstream variability. The Combinati SARS-CoV-2 Wastewater Surveillance 4-plex assay was specifically designed to quantity 2 SARS-CoV-2 viral targets (N1 and N2) alongside a human fecal normalization control (PMMoV) and the sample process control (BCoV) in a single digital PCR reaction. This enables 4x the amount of data to be collected per sample run than many traditional qPCR reactions. Using four separate color channels enables simplified detection of each target without the need for multiple standard curves or tedious manual gating of multiple positive clusters per channel.
This technical note highlights how the sample process control Bovine Coronavirus (BCoV) can be utilized to provide information regarding the success and efficiency of nucleic acid extraction without impacting the sensitivity or accuracy of SARS-CoV-2 detection and quantification.

Using a process control to evaluate sample extraction efficiency

Input Considerations

A spike in process control is intended to provide quantitative information about sample loss during sample processing workflow. Typically, a process control is spiked into the initial sample before any processing takes place, and the presence of that control at the end of the workflow serves as an indicator of successful sample recovery.
For the SARS-CoV-2 Wastewater Surveillance 4-plex assay, the endpoint readout of the Bovine Coronavirus target (BCoV) is an absolute quantification of the total number of molecules, determined through digital PCR. By quantifying the control material both prior to spike in and after the processing workflow is complete, an independent measure of the overall yield of the workflow can be calculated. This is enables accurate evaluation of workflows without depending on endogenous targets that could naturally vary from sample to sample.

Interpreting Digital PCR Data

Results reported by the Absolute Q Analysis software are the concentration of each target in copies per microliter (cp/µL) in each dPCR reaction. To calculate the concentration of targets in the original input sample, first calculate the total number of molecules in the dPCR reaction by multiplying the concentration by the 9µL total volume of the dPCR reaction (Equation 1).
Next, to calculate the concentration of the sample used as input into the dPCR reaction, divide the quantity calculated using Equation 1 by the volume of sample used as input. For example, if 5µL of extracted RNA from wastewater was used per dPCR reaction, divide the quantity by 5µL to obtain the original extracted RNA concentration.

Evaluating workflow efficiency

Comparing the concentration of BCoV spiked into the original wastewater sample to the amount of BCoV recovered post processing can provide insight into the overall yield of the workflow. For specific suggestions on how to quantify and use BCoV as a wastewater sample process control consult the SARS-CoV-2 Wastewater Surveillance Kit Instructions for use.

Using Bovine Coronavirus as a process control for wastewater SARS-CoV-2 monitoring

Here we highlight the consistent quantification of BCoV using an example sample extraction workflow. After reconstituting the BCoV control material (materials and methods), 5 serial 10-fold dilutions were prepared, extracted and quantified using the SARS-CoV-2 Wastewater Surveillance Kit. In addition to the BCoV serial dilutions, a SARS-CoV-2 positive reference material and water-only conditions were included as positive and negative extraction controls.

All samples were tested using the SARS-CoV-2 Wastewater Surveillance 4-plex Kit. Figure 1 highlights the consistency of quantification between replicates as well as linearity of the serial dilutions of BCoV (p<0.001). The results suggest that the RNA extraction was successful and the quantification was consistent across the range of input material (Figure 1A). The positive and negative process controls as well as PCR control, which included controls for all 4 assay targets, behaved as expected (Figure 1B-D).

To verify the BCoV concentration obtained from the multiplex assay, a simplex BCoV assay was used to quantify the same purified RNA samples. The average concentration of each sample from the simplex assay showed high is completely in concordance with the concentration from the multiplex assay with a significant Pearson correlation (R2 = 1.0, P<0.0001) Table 1.

Calculating input concentration by dPCR

Using the dPCR data, concentration of the original BCoV material can be calculated using Equation 1. Since one microliter was used as input into each PCR reaction, concentration of the original material can be calculated by accounting for the initial dilution factor for sample extraction. Based on the results, the concentration of the original BCoV input material prior to extraction is 2.39E+06 (Table 2).

Accuracy and sensitivity for SARS-CoV-2 targets remain high

To demonstrate the sensitivity and accuracy of the 4-plex assay in the presence of spike in process control material, we validated the quantification accuracy of the 4-plex assay using three levels of SARS-CoV-2 target concentrations with backgrounds of 5-log concentration range of BCoV (materials and methods).
Three concentrations of SARS-CoV-2 reference RNA (stock , 10-fold and 100-fold dilutions) were added to each point of the BCoV extracted RNA dilution series along with a constant quantity of PMMoV ssDNA (materials and methods). Each mixed sample was then tested using the SARS-CoV-2 Wastewater Surveillance 4-plex assay. The 3 serial 10-fold SARS-CoV-2 reference RNA control materials were also tested without BCoV and PMMoV to evaluate the effects of the spike in material on quantification.
As shown in Figure 2, while the BCoV concentration decreases with each serial dilution point, the quantity of the N1 and N2 remain constant for the stock concentration (Figure 2a), 10-fold dilution (Figure 2b) and 100-fold dilution (Figure 2c) of SARS-CoV-2 reference material. The level of PMMoV (red-bars) remained constant for all conditions tested and yielded a concentration of 46.37 copies/µL (±4.78cp/µL) across all 47 reactions. The measured concentrations with and without BCoV spike-in RNA in the reactions are highly correlated for both N1 and N2 targets as shown in Figure 2d.

Workflow/Materials and Methods

Nucleic Acid Material

Extracted RNA, synthetic construct and standard reference materials were used in the experiments. The Bovine Coronavirus RNA has been extracted from the modified live virus BOVILIS CORONAVIRUS (Merck Animal Health). A 10-dose was reconstituted in 2 milliliters 1x TE buffer (Thermo Fisher Scientific, Waltham, MA) and serially diluted to 1:10, 1:100, 1:1,000, 1:10,000 and 1:100,000. RNA extraction was performed on Maxwell RSC 16 (Promega, Madison, WI). 50 µL of each dilution were extracted along with extraction controls including nuclease free water as negative control and positive control AccuPlex SARS-CoV-2 Positive Reference Material (SeraCare, Milford, MA). All samples were eluted in 50 µL nuclease free water and stored in -80ºC freezer. Additionally, single stranded synthetic DNA for Pepper Mild Mottle Virus (PMMoV) and Exact Diagnostic SARS-CoV-2 RNA control material (Bio-Rad, Hercules, CA) with three dilutions (stock, 1:10 and 1:100) were used in the digital PCR experiments.
Two hydrolysis probe-based assays were used in the study. 

Combinati SARS-CoV-2 Wastewater Surveillance 4-plex Kit

The Combinati 4-plex SARS-CoV-2 Wastewater Surveillance assay specifically detects SARS-CoV-2 N1 (FAM) and N2 (HEX) as well as the targets for BCoV process control (TAMRA) and PMMoV human fecal normalization control (TYE) in a single multiplexed reaction. A simplex assay with a probe in the TAMRA channel was used to measure the extracted BCoV RNA.
After preparing the one step RT-dPCR mix, 9µL of the reaction mixture was loaded into the MAP16 plate followed by an overlay of 15µL of isolation buffer. The prepared MAP16 plate was then loaded on the Absolute Q. Figure 3 details the thermal cycling and reagent preparation protocols for RT-dPCR on the Absolute Q. Following the RT-dPCR, the sample concentrations were determined using the Absolute Q Analysis Software with the following threshold for each target FAM, 5000 fluorescence units, HEX 1000 fluorescence units, TAMRA 1300 fluorescence units). Statistical Analyses were performed using Graphpad Prism 9.1.0 (La Jolla, CA)

References

Wu, Fuqing, et al. “SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases.” MSystems, vol. 5, no. 4, 2020, doi:10.1128/msystems.00614-20.

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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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.

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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/.

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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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/.

Discriminatory 11-Plex Assay Designed to Monitor Estrogen Receptor Mutations Using a Novel, Melt-Analysis Capable Digital PCR Platform​

Discriminatory 11-Plex Assay Designed to Monitor Estrogen Receptor Mutations Using a Novel, Melt-Analysis Capable Digital PCR Platform​

In a large fraction of ER+/HER2- metastatic breast cancer (MBC), treatment with aromatase inhibitors fails due to emerging resistance. A key mechanism of this resistance is associated with a set of missense mutations in the gene encoding for the estrogen receptor (ESR1).

Digital PCR has exceptional sensitivity for detecting rare targets, making it ideal for detection of mutations linked to drug resistance. However, standard TaqMan-based assays rely upon competitive probe dynamics to maintain SNP-target specificity in mixed samples.  This poster highlights the melt-capable Combinati Absolute Q digital PCR system to facilitate higher-order multiplexing to  quantify more alleles in less time using less input sample required to achieve the same results by other assay methods.

Here we showcase a multiplex, quantitative, Research Use Only assay for the detection and identification of 11 ESR1 single nucleotide polymorphisms (SNPs) and the ESR1 gene in a single reaction, using patented, melt-based chemistry and an innovative digital PCR (dPCR) system.

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4-color Copy Number Variation (CNV) Assay for Spinal Muscular Atrophy (SMA) Screening

Background

Spinal muscular atrophy (SMA), a genetic neuromuscular disorder and historically a leading genetic cause of infant mortality, is largely caused by a copy number variation event resulting in the loss of the survival motor neuron 1 (SMN1) gene. The duplicate SMN gene, survival motor neuron 2 (SMN2), has been found to produce partial function and can partially compensate for SMN1 deletion and reduce SMA disease severity. Accurate and timely quantification of SMN1 and SMN2 copy number variation provides critical diagnostic and prognostic values for the disease. Here we describe a 4-color multiplexing digital PCR (dPCR) solution using the Combinati Absolute Q dPCR system to meet the growing demand for rapid SMA newborn screening and treatment decisions.

Figure 1. Schematic view of the 4-color multiplex SMA assay design Copy Number Variation
Figure 1. Schematic view of the 4-color multiplex SMA assay design

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