Application Notes: Absolute Quantification

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, the reaction mixture was loaded into the MAP16 plate followed by an overlay 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 

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

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.

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