Application Notes: Tech Note

Improve sensitivity for rare targets by increasing total analyzed sample using digital pooling of digital PCR data

Introduction

A primary application of digital PCR (dPCR) is rare target detection. Absolute quantification, rather than cycle threshold values enable users to obtain the count of original target molecules in a sample – eliminating the need for interpreting amplification curves or relying on reference material to provide quantitative measurements. This improves both accuracy and reproducibility of concentration measurements of rare targets.
However, the limit of detection for any digital PCR reaction is dependent on several factors. These include the concentration of the target of interest and the volume of sample that can be loaded per reaction (Figure 1). In scenarios where the target of interest is present at a very low concentration, it may be necessary to run more than one reaction in order to sample enough volume to detect the target.

One example of rare target detection is the measurement of circulating tumor DNA (ctDNA) among normal circulating cell free DNA molecules (ccfDNA) from liquid biopsy samples of individuals diagnosed with cancer. In this application, the total number of ctDNA molecules can vary from patient to patient as well as from day to day; and are often very low in concentration – typically only several molecules per microliter. If the concentration of ctDNA molecules is exceptionally low, the amount of sample tested will directly impact the number of ctDNA molecules that can be detected. Furthermore, many existing digital PCR platforms suffer from lack of consistency and reproducibility in the total number of partitions generated due to limitations in the underlying partitioning technology, resulting in unreliable detection. This inconsistency can cause variability in the total amount of sample analyzed per reaction as well as between experiments. The wasted sample can contribute to sub-sampling error. Unlike emulsion based dPCR platforms, the microfluidic array partitioning (MAP) technology used in Absolute Q dPCR partitioning is entirely automated and highly consistent – utilizing an industry leading 95% of loaded sample volume across over 20,000 partitions each time.
This technical note highlights the digital pooling feature of the Combinati Absolute Q by leveraging an Applied Biosystems® TaqMan™ Liquid Biopsy Assay for the cancer mutation PIK3CA p.H1047R (cat. A44177). A contrived sample with mutant allele fraction at 0.1% was created and tested and digitally pooled across 4 dPCR reaction arrays (~81,920 partitions) .

Download PDF Technical Note

Workflow and Methods

DNA Mixture and dPCR reaction preparation

Using genomic DNA (Promega Human Male Control) and plasmid DNA containing the PIK3CA p.H1047R mutation, a DNA mixture of 0.1% MAF was prepared with final human genomic DNA concentration of approximately 1.15 ng/µL. The Applied Biosystems TaqMan Liquid Biopsy assay for PIK3CAp.H1047R which detects both the wild type (VIC) and mutation (FAM) alleles was used in this study. The digital PCR reactions were prepared according to the volumes indicated in Table 1.

Digital PCR reagent table

Reagent Preparation and Digital Pooling


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. The prepared MAP16 plate was then loaded on the Absolute Q. Standard thermal parameters for use of the Applied Biosystems® TaqMan™ Liquid Biospy Assays on Absolute Q were used. Following the dPCR run, target concentrations were determined using the Absolute Q Analysis Software. A total of eight reactions were run using the samples and subsequently four arrays each were digitally pooled for a total of two replicates.
As an orthogonal test, a positive control at higher concentration of 0.1% MAF DNA mixture was prepared using the same plasmid and human genomic DNA. This DNA sample was used for 2 independent replicates and loaded at a final concentration of 33ng per reaction or approximately 10,000 wild type molecules and 10 mutation molecules per reaction.

Results

After dPCR was complete, all reactions were analyzed using the Absolute Q Analysis software. The dPCR results for the pooled low concentration 0.1% MAF sample across 4 arrays and high positive concentration control are shown in Table 2. Both conditions reported concentrations of the mutation and wild type PIK3CA molecules similar to the expectations. As indicated in Table 2, across approximately 81,545 digitally pooled partitions, the PIK3CA mutation allele was calculated to be at a concentration of 11.5 copies per reaction (cp/reaction) – similar to the positive control reaction which was determined to be 6.9 cp/reaction. Finally, the observed MAF of the digitally pooled low concentration sample was 0.1% MAF as expected.

Summary

Digital pooling is an effective method to increase sensitivity by increasing the amount of volume that can be analyzed for a given sample. In this technical note, four arrays (36µL dPCR Mix) of the MAP16 plate were used to analyze a total input sample volume of 28.8µL of a 0.1% MAF DNA mixture. Overall this method has the potential to be applied to a multitude of rare target detection applications in the precision medicine space such as monitoring treatment response, screening for minimal residual disease or rapid identification of mutations linked drug resistance.

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.

Download PDF

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.

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.

If you’d like to print this page, click the button below to download a PDF version of this technical note.

Download PDF

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.

If you’d like to print this page, click the button below to download a PDF version of this technical note.

DOWNLOAD THE APP NOTE

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.

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.

If you’d like to print this page, click the button below to download a PDF version of this technical note.

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

If you’d like to print this page, click the button below to download a PDF version of this technical note.

Download PDF
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

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

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.

Applied Biosystems™ TaqMan® Liquid Biopsy Assays for Rare Target Quantification

Introduction

Mutation screening is becoming a standard for evaluating treatment options of patients diagnosed with cancer. Notably, certain hotspot mutations can give valuable insight into efficacy of response to various treatments. For example, mutations such as EGFR p.T790M and KRAS p.G12D, p.G12V and p.G13D indicate potential reduced responsiveness to Tyrosine Kinase Inhibitors (TKIs), whereas PIK3CA mutations such as p.H1047R indicate positive response to PI3K/AKT/mTOR signaling pathway inhibitors. With its unparalleled precision and sensitivity, digital PCR is ideally suited for liquid biopsy applications in which low amounts of relevant mutations exist in the sample.

The Combinati Absolute Q, a novel one-step dPCR technology, was used with the Applied Biosystems™ TaqMan™ Liquid Biopsy dPCR Assays to perform rare target detection for 5 hot-spot cancer mutations in the KRAS, EGFR, and PIK3CA genes. MAFs down to 0.1% were detected among a high background wild-type concentration.

If you’d like to print this page, click the button below to download a PDF version of this technical note.

Download Application note

Workflow and Methods

Titration Series Preparation

Five hot-spot cancer mutations were selected for this study: KRAS p.G12D, KRAS p.G12V, KRAS p.G13D, EGFR p.T790M, and PIK3CA p.H1047R. A series of DNA mixtures were prepared by titrating mutation-bearing plasmids into a high background of wild-type normal gDNA for each of the mutations selected. Each point along the titration series contained 15 nanograms of gDNA, and mutation plasmid targeting final mutation allele frequencies (MAFs) of 10%, 1%%, 0.1%. and 0%.

Absolute Q Digital PCR Workflow for pcr machine

Figure 1. Absolute Q workflow.

Absolute Q Workflow

Using the Absolute Q’s simple workflow, PCR mix was prepared using the Combinati 5X MasterMix and loaded into the microfluidic array partitioning (MAP) plate. Subsequently, 10µL of Combinati Isolation Buffer was overlaid into each well used and gaskets applied across all units of the plate. Finally, the plate was loaded onto the Absolute Q where partitioning, thermal cycling, and data collection were completed on the instrument in approximately 90 minutes.

The thermal cycling parameters for use of the TaqMan Liquid Biopsy dPCR Assays on the Absolute Q are listed in Table 1. For select performance evaluation studies, the extension temperature and thermal dwell times were modified. Modifications are indicated when applicable.

Table 1. Thermal Cycling Parameters on the Combinati Absolute Q.

Table 1. Thermal Cycling Parameters on the Combinati Absolute Q.

Results

Rare Mutation Allele Detection

Detecting circulating tumor DNA for liquid biopsy applications is challenging because the molecules bearing the target of interest are only a small fraction of the total circulating cell free DNA collected in the sample. Liquid biopsy assays must be able to accurately quantify rare, single nucleotide polymorphisms, among high levels of wild-type background DNA with outstanding sensitivity and precision. For this study, five cancer-relevant mutations were selected to demonstrate the high precision and sensitivity on the Absolute Q (Workflow and Methods).

For each assay, the total number of mutation molecules and observed mutation allele fraction (MAF) were calculated for each point of the titration series (10%, 1%, 0.1% and 0%). Figure 2a illustrates 2-dimensional digital PCR data generated on the Absolute Q from one replicate of each titration point of the KRAS p.G12V assay test. As expected, the number of total mutation (FAM channel, denoted in blue) positive partitions decreases with each successive point, reflecting a reduction in KRAS pG12V mutation molecules overall while the wild-type (VIC channel, denoted in red) partitions remain constant. Figure 2b shows the linear relationship observed between the expected and observed MAF across all titration series assays. Using a Pearson correlation test, the total number of observed mutation molecules and calculated MAF for each of the titration series were highly correlated with the expectations for all five assays (R = 1.0, p<0.001).

Figure 2A. Two dimensional scatter-plot representation of individual partition amplitudes in the VIC and FAM channels of the Combinati Absolute Q for the KRAS p.G12V specific assay.

Figure 2A. Two dimensional scatter-plot representation of individual partition amplitudes in the VIC and FAM channels of the Combinati Absolute Q for the KRAS p.G12V specific assay. 

Figure 2B. Results of absolute quantifitacion of mutation molecules across a titration mutation bearing plasmid spiked into a background of 15 nanograms of wild-type human genomic DNA. Titration series points were targeted to be 10%, 1% and 0.1% mutation allele frequency (MAF).

Figure 2B Results of absolute quantifitacion of mutation molecules across a titration mutation bearing plasmid spiked into a background of 15 nanograms of wild-type human genomic DNA. Titration series points were targeted to be 10%, 1% and 0.1% mutation allele frequency (MAF).

5X Digital PCR Mix for Low Concentration Samples

A major challenge for many liquid biopsy applications is detection of ultra-rare variants which exist at an extremely low concentration. Sample input volume alongside workflow dead-volume can inhibit the detection of these rare variants by limiting the total number of accessible molecules possible for input. To address this, Combinati offers a 5X dPCR MasterMix for use with liquid biopsy assays. Use of this formulation enables users to add up to 66% more sample volume to each dPCR reaction than when using a 2X formulation. We compared the performance of the 5X MasterMix against a standard 2X PCR MasterMix – testing each assay’s full titration series. Both formulations demonstrate comparable quantification results across each titration series (Figure 3).

Figure 3. Comparison of 5X Combinati mastermix (purple) and 2Xmastermix (orange) mutation molecule quantification for 5 liquid biopsy assays.

Figure 3. Comparison of 5X Combinati mastermix (purple) and 2Xmastermix (orange) mutation molecule quantification for 5 liquid biopsy assays.

Best-in-class Reagent Partitioning with the MAP16

The Combinati Absolute Q utilizes a novel method of reaction isolation on fixed microfluidic array partitioning (MAP) plates. Without a reliance on emulsion based reagent partitioning, over 90% of the loaded sample is analysed in each unit, improving confidence in rare molecule detection. The MAP16 plate consistently generates approximately 20,000 of uniformly filled micro-reactions with no user interaction. The total number of viable partitions across 5 plates are shown in Figure 4. With the exception of one, each unit had over 20,300 viable partitions with an average of 20,457 (±98) accepted partitions per unit.

Figure 4. Total accepted or viable partitions of each of the 16 units per plate for 5 MAP plates used for the Liquid Biopsy assay testing.

Figure 4. Total accepted or viable partitions of each of the 16 units per plate for 5 MAP plates used for the Liquid Biopsy assay testing.

Figure 5. Comparison of ultra-fast and standard dPCR thermal protocols on Absolute Q.

Figure 5. Comparison of ultra-fast and standard dPCR thermal protocols on Absolute Q.

Short Dwell Times Maintain High Specificity

The Absolute Q dPCR platform paired with the MAP plate enables targeted thermal points to be reached much quicker, due to the low reagent volume sequestered in each partition. Using this feature, the dwell times at denaturation and annealing extension were set to 0 seconds, thereby reducing the overall time to complete PCR. To demonstrate heightened precision possible using ultra-fast PCR, we compared our dPCR thermal protocol listed in Table 1 (0 second denature, 0 second annealing and extension) to the current dPCR protocol recommendation of 15 seconds denature, 30 seconds anneal/extend. We performed this test using the PIK3CA p.H1047R titration. Figure 4 shows consistent quantification performance for low MAF targets between the two thermal profiles.

Summary

Digital PCR (dPCR) enables rare target detection even among a high amount of background non-target presence. The Combinati Absolute Q dPCR platform consists of a familiar microtiter-plate formatted consumable and a fully integrated instrument. The platform offers a simplified one-step workflow identical to traditional quantitative PCR (qPCR). The Absolute Q was used with the Applied Biosystems TaqMan Liquid Biopsy dPCR Assays to perform rare target detection for 5 hot-spot cancer mutations. A sensitivity of 0.1% MAF was demonstrated for each assay showcasing the Absolute Q’s cross-assay robustness for clinically relevant rare target detection.

More to Download