Application note | TrueMark Infectious Disease Custom Research Panels

Clinical research

Direct PCR: enabling an extraction-free

workflow with off-the-shelf reagents
for a more efficient testing procedure, especially in clinical
In this report, we highlight that direct PCR, an research settings where cost and time are key. Direct-to-PCR
extraction-free workflow: or direct PCR is an alternative workflow where the sample
• Can serve as an alternative to an extraction-based workflow bypasses extraction for downstream PCR, streamlining existing
for simpler, streamlined processing, specifically of infectious workflows. To demonstrate the potential value and tradeoffs
respiratory targets of direct PCR, direct PCR was evaluated across four common
• Can be used with commercially available products, including respiratory pathogens—SARS-CoV-2, adenovirus, influenza A,
TrueMark Infectious Disease Custom Research Panels and influenza B—using nasal swabs, which are easily obtained

• Can tolerate sample heterogeneity and perform across

research specimens.
multiple transport media
Workflow
Direct PCR replaces a multistep nucleic acid extraction workflow
with a faster and simpler lysis step that requires only heat and
Introduction
two readily available reagents. In contrast, traditional nucleic
While PCR is the backbone of many molecular testing
acid extraction methods require significant hands-on time to
applications, including those involving infectious pathogens, PCR
prepare reagents as well as additional plastics and equipment.
typically requires an input of extracted nucleic acids. Prior to
Furthermore, the reagents and consumables in common nucleic
PCR, an extraction workflow isolates, purifies, and concentrates
acid extraction steps—lysis, nucleic acid binding, washing,
nucleic acids from the input sample matrix. However, extraction
and elution—generate significant amounts of plastic and
requires hazardous chemicals, tedious hands-on time, dedicated
hazardous waste, further burdening the lab with additional costs.
instruments, and additional costs for each sample. Hence,
Thus, direct PCR presents multiple workflow savings as depicted
there has been growing interest in eliminating extraction
in Figure 1.

MagMAX Proteinase K
Figure 1. Direct PCR vs. nucleic acid
A. Direct PCR extraction. (A) With a direct PCR method,
workflow Treated sample
samples are loaded with two reagents and
+ heat-treated at 95°C for 5 minutes prior to
Carrier RNA
Sample treatment
PCR. (B) Nucleic acid extraction workflows
(95°C incubation, can be performed with the Applied
~5 min per plate)
Biosystems™ MagMAX™ Viral/Pathogen II
Nucleic Acid Isolation Kit on the Thermo
Scientific™ KingFisher™ Flex instrument as a
RT-PCR amplification semi-automated solution.
Sample and data analysis

B. Extraction Purified nucleic

workflow acid
Nucleic acid extraction
Sample, extraction reagents, (~23 min* per plate)
and plate peparation
(~15 min per plate)

* Time stated is based on the protocol described in the user guide [1].
Here we compare direct PCR to a common extraction-based Table 2. Assay and target information for the TrueMark
workflow that leveraged the MagMAX Viral/Pathogen II Nucleic Infectious Disease Custom Research Panels.
Acid Isolation Kit on the KingFisher Flex instrument, to evaluate Target (label) Assay ID Label (quencher)
their relative performance in research applications to detect Vi07922136_s1
Adenovirus* FAM (QSY)
common respiratory pathogens from contrived nasal swabs. Vi07922137_s1
Influenza A** Vi99990011_po FAM (MGB)
Direct PCR sample preparation workflow
For direct PCR, the sample, Applied Biosystems™ MagMAX™ Vi07922143_s1
Influenza B* FAM (QSY)
Vi07922144_s1
Viral/Pathogen Proteinase K (Cat. No. A42363), carrier RNA
SARS-CoV-2** Vi07921935_s1 FAM (MGB)
(Cat. No. 4382878), and nuclease-free water were mixed and
heat-treated for 5 minutes at 95°C as described in Table 1. This RNase P †
CDC RNase P [2] JUN (QSY)
treatment assists in pathogen lysis, releasing nucleic acid for * 300 nM primers, 250 nM probes.

downstream PCR, matrix compatibility with PCR, and protection ** 900 nM primers, 250 nM probes.

of nucleic acids from degradation. The heating step can leverage † 300 nM primers, 150 nM probe [2].

an existing PCR thermal cycler or a heat block.

To perform RT-PCR, a sample (direct PCR or extracted) along
Table 1. Sample treatment reaction setup. with Applied Biosystems™ TaqPath™ 1-Step Multiplex Master Mix
Final (No ROX) (Cat. No. A28523) and nuclease-free water was loaded
Component* Per well concentration into the pre-spotted PCR plate containing the dried assay, as
Sample 90 µL 90% shown in Table 3. To verify amplification performance, positive
MagMAX Viral/ and negative controls were included in all experiments.
Pathogen Proteinase K 5 µL 2.5 mg/mL
(50 mg/mL) Table 3. RT-PCR reaction setup for extraction and direct
Carrier RNA (1 mg/mL) 4 µL 0.04 mg/mL PCR workflows.
Nuclease-free water 1 µL – Volume per well
Total volume 100 µL – Extraction Direct PCR
* Optimal sample treatment conditions may be specific to the pathogen or sample type; lysis was Component workflow workflow
performed in a 96-well, 0.2 mL PCR plate on a thermal cycler or heat block.
Master mix (4X) 5 µL 5 µL
Nuclease-free water 10 µL 5 µL
Extraction sample preparation workflow
Sample input 5 µL 10 µL
For comparison to direct PCR, an extraction-based workflow was
Total volume 20 µL 20 µL
used for benchmarking. Nucleic acid extraction was performed
with the MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit on All testing discussed here was performed on the Applied
the KingFisher Flex instrument using a 200 µL sample input and Biosystems™ QuantStudio™ 5, 96-well, 0.2 mL Real-Time PCR
50 µL of elution buffer. System and analyzed using Applied Biosystems™ QuantStudio™
Design and Analysis Software (v2.6).
Detection of respiratory pathogens and
endogenous control The thermal cycling profile was found to be a key parameter for
The evaluated respiratory targets were detected using Applied the success of direct PCR, as direct PCR requires the reaction
Biosystems™ TaqMan™ Array Plates, specifically the Applied to tolerate a wide range of inhibitors and concentrations of
Biosystems™ TrueMark™ Infectious Disease Custom Research reaction components, such as salts. To enable more tolerant,
Panel (Cat. No. A55017), which are pre-spotted with dried-down robust performance across a broader number of input materials,
assays that detect SARS-CoV-2, adenovirus, influenza A, and it was found that subtle changes in thermal profiles were critical
influenza B viruses, each duplexed with human RNase P (RNase to performance for direct PCR. Based on these observations,
P), which serves as an endogenous sample control (Table 2). a cycling profile specific to direct PCR was optimized for use
These plates are custom ordered, allowing customers to select with the TaqPath 1-Step Multiplex Master Mix (No ROX), and a
the assays and plate layout best suited to fit their needs. standard, recommended cycling profile was leveraged for the
extraction workflow.

2
The respective cycling profiles can be found in Table 4. For analysis in the
Applied Biosystems™ Design and Analysis Software following the PCR run, a baseline
threshold of 10,000 was applied to all targets, and detection of a Cq of <40 was
leveraged for each target. Note that test results should be analyzed according to the
analysis, interpretation, and QC parameters validated by your laboratory.

Table 4. Thermal cycling profiles for extraction and direct PCR workflows.
Workflow Step Temperature Time Number of cycles Ramp rate
UNG incubation 25°C 2 min 1
Reverse transcription 53°C 10 min 1
Preincubation 85°C 10 min 1 1.6°C/sec for heating up and
Extraction
Activation 95°C 2 min 1 cooling down
Denaturation 95°C 3 sec
40
Anneal/extension 60°C 30 sec
Reverse transcription 53°C 5 min 1
Activation 95°C 1 min 1
1.91°C/sec for heating up;
Direct PCR Denaturation 95°C 2 sec
1.64°C/sec for cooling down
Annealing 60°C 8 sec 42
Extension 68°C 25 sec

Performance
Comparison of analytical sensitivity of workflows
Table 6. Quantified test materials.
When evaluating direct PCR and nucleic acid extraction, one
Organism Genome Source Cat. No.
area where extraction-based workflows have an advantage is
SARS-CoV-2,
the target concentration when a large-volume sample (such RNA BEI Resources NR-52287
USA-WA1/2020
as 200–400 µL) is taken, concentrated, and eluted (typically
Influenza A, H1N1
50–100 µL) for PCR; Table 5 highlights typical concentration RNA ZeptoMetrix 0810244CF
Brisbane/59/07
factors in extraction-based workflows. PCR reaction volume and
Influenza B,
amount of sample or eluate loaded into PCR can further influence RNA ZeptoMetrix 0810517CF
Victoria/2/87
the concentration factor. Based on the workflows evaluated here,
Adenovirus Type
the extraction-based workflow theoretically loads approximately DNA ZeptoMetrix 0810021CF
7A
4x as much sample as in the direct PCR workflow.
The impact of workflow on respiratory pathogen detection was
Table 5. Common extraction-based workflow estimated by testing a dilution series in both the extraction and
concentration factors. direct PCR workflows (Table 7). The lowest level at which 100%
Elution volume detection was obtained for each workflow was determined.
Input sample volume 50 µL 100 µL Varying with target, a 3x to 9x difference in relative sensitivity was
200 µL 4x* 2x observed; this is in line with the estimated theoretical difference
400 µL 8x 4x calculated (Table 5, 4x). Depending on the application and needs
* Theoretical sample concentration factor in the evaluated workflow. of a user (cost, turnaround time), the tradeoff in sensitivity may
be acceptable.
Thus, when considering a direct PCR workflow, it is important
to consider the sensitivity needs of the application. To quantify
sensitivity differences across respiratory pathogens (Table 6),
a sample matrix of nasal swabs suspended in viral transport
medium (VTM) [3] was used.

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Table 7. Sensitivity of direct PCR and extraction-based workflows.
Detection rate (10 replicates)Sensitivity:
extraction vs. direct
Target Sample concentration Extraction workflow Direct PCR workflow PCR
2 100% 100%
0.67 100% 100%
Adenovirus type 7A 0.22 100% 50%
3x
(TCID50/mL*) 0.074 90% 10%
0.025 50% 10%
0.0082 20% 0%
1650 100% 100%
550 100% 90%
SARS-CoV-2 180 100% 70%
9x
(GCE/mL*) 60 80% 40%
20 40% 0%
7 30% 10%
0.3 100% 100%
0.1 100% 90%
Influenza A
0.033 100% 30%
H1N1 Bris/59/07 9x
0.011 50% 10%
(TCID50/mL)
0.0037 30% 0%
0.0012 10% 0%
0.15 100% 100%
0.05 100% 100%
Influenza B
0.017 100% 90%
Victoria/2/87 3x
0.0056 90% 60%
(TCID50/mL)
0.0019 60% 80%
0.00062 40% 10%
* TCID50: 50% tissue culture infectious dose; GCE: genome copy equivalents.

Direct PCR workflow robustness among common transport media, the robustness of direct PCR
Nucleic acid extraction involves purifying nucleic acids from an to both of these factors was evaluated by spiking with SARS-
input sample’s matrix and isolating them in a specific elution CoV-2 and adenovirus at 3x the established limits of detection
buffer. In contrast, direct PCR incorporates all components from previously determined and shown in Table 7.
the sample matrix directly into the PCR reaction without prior
Workflow robustness across individual samples
purification. Thus, direct PCR is exposed to higher variation due
Across ten individual nasal swab samples, direct PCR was able
to sample-to-sample heterogeneity and sample transport media.
to detect all spiked pathogens and RNase P from each donor
To assess the impact of both sample heterogeneity and variation
(Table 8) demonstrating robustness to sample heterogeneity.
Table 8. Direct PCR target detection across individual samples.
Spiked pathogen detection in negative nasal dry-swab samples (VTM)
Adenovirus Swab ID 1 2 3 4 5 6 7 8 9 10
type 7A Detection 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%
(2 TCID50/mL) Cq 33.6 33.46 33.27 33.61 35.65 34.81 33.6 34.02 33.33 33.89
Swab ID 1 2 3 4 5 6 7 8 9 10
SARS-CoV-2
Detection 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%
(4,950 GCE/mL)
Cq 32.26 32.52 31.79 32.46 31.09 31.4 31.95 32.25 31.46 32.41
RNase P detection in negative nasal dry-swab samples (VTM)
Swab ID 1 2 3 4 5 6 7 8 9 10
RNase P
Cq 31.56 28.42 27.06 29.19 21.03 26.13 31.87 31.99 29.58 30.24

4
Workflow robustness across transport media
Similarly, we evaluated the robustness of direct PCR to two alternative commonly
used transport media, Universal Transport Medium™ system (Copan Diagnostics) and
Thermo Scientific™ MicroTest™ M4RT medium. Pathogen targets and RNase P were
successfully detected in all sample replicates, as shown in Table 9.

Table 9. Direct PCR target detection across transport media.

Transport medium Spiked pathogen detection in negative nasal dry-swab samples
Detection (detected/tested) 100% (10/10)
Adenovirus type 7A
Universal Transport Cq (SD) 37.74 (0.28)
Medium system Detection (detected/tested) 100% (10/10)
SARS-CoV-2
Cq (SD) 31.31 (0.21)
Detection (detected/tested) 100% (10/10)
Adenovirus type 7A
Cq (SD) 31.22 (0.43)
MicroTest M4RT medium
Detection (detected/tested) 100% (10/10)
SARS-CoV-2
Cq (SD) 32.08 (0.26)
Transport medium RNase P detection in negative nasal dry-swab samples
Universal Transport Detection (detected/tested) 100% (20/20)
RNase P
Medium system Cq(SD) 28.10 (0.12)
Detection (detected/tested) 100% (20/20)
MicroTest M4RT medium RNase P
Cq (SD) 28.99 (0.21)

While all targets were detected with direct PCR, slight differences • Accessible: Direct PCR can be performed with readily
in Cq values highlight the importance of characterizing the specific available reagents and instruments, based on the workflow
use case, collection media, targets, and assays when running described here.

direct PCR. However, the subset tested here demonstrated • Respiratory pathogen applications: For research uses,
robustness to nasal swab heterogeneity and transport media, direct PCR can robustly detect selected pathogens from nasal
supporting direct PCR as a potential streamlined workflow swabs, performing across multiple samples and transport
media.
solution for molecular testing.

Conclusions References
1. MagMAX Viral/Pathogen II Nucleic Acid Isolation Kit, User Guide (MAN0024756)
Direct PCR presents a potentially advantageous alternative to the
2. cdc.gov/coronavirus/2019-ncov/lab/multiplex.html
conventional extraction-based workflow, particularly in the realm
3. cdc.gov/coronavirus/2019-ncov/downloads/Viral-Transport-Medium.pdf
of respiratory infectious disease testing.

• Simplified workflow: Direct PCR reduces the need for nucleic

acid isolation, reducing turnaround time, hands-on time,
and cost.

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