1

Navigating amplification
workflows for robust
diagnostic assay development
Introduction
Knowledge of amplification techniques and optimization tactics is crucial to driving
innovation in molecular diagnostics (MDx). During the COVID-19 pandemic, the number of
molecular diagnostic assays incorporating amplification technologies increased significantly
to meet public health needs. This expansion was enabled by years of workflow optimization,
broader access to enhanced reagents and design tools, and the deployment of common
equipment and automation worldwide. Molecular diagnostic workflow improvements led to
substantial time savings and critical quality metrics – from the point of sample collection to
the result. With diligent planning, the accuracy, sensitivity and specificity of an assay can be
effectively established for the successful implementation of any molecular diagnostic.

The pandemic also broadened the scope of amplification methods validated for sensitive
and robust molecular diagnostic tests, putting isothermal protocols into use alongside qPCR
(the ‘gold standard’). Isothermal amplification methods, such as Loop-mediated Isothermal
Amplification (LAMP) were adopted for their simplicity and speed, which permitted at-home
testing. The ability to collect a sample, perform a molecular assay, and read the results at home
offered many advantages over traditional testing scenarios. The standard testing paradigm
required a visit to a hospital or clinic for sample collection, after which the test itself was
performed in a central laboratory. Subsequently, a healthcare provider would contact the
patient with the results. This process could take several hours, or even days, before the results
were available for clinical decision-making. If instead tests and results could be provided at
the point-of-care or even at home, this timeline could be accelerated significantly, and these
efforts have necessitated a shift to supply reagents as lyophilized or dry versions of the assays.
These efforts are leading to an increased demand for lyophilized formats.

Despite these advances, many goals remain the same for MDx assay developers: determining
the appropriate amplification method, optimizing assay components, and selecting a reliable
supply partner who will provide support from early development all the way through to large-
scale manufacturing.

Learn more about products that

can be used in the development
and manufacturing of your
diagnostic and therapeutic at
[Link]/innovation

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Nucleic acid amplification-based
diagnostics pipelines
Nucleic acid amplification tests (NAATs) are used extensively for in vitro Use Authorization (EUA) during public health emergencies, such as
human diagnostics disease screening and detection and have also found the COVID-19 pandemic, which allow medical tests to diagnose
utility in other areas, such as veterinary and agricultural testing. NAATs and treat serious diseases for which no adequate or alternative
employ a DNA polymerase to amplify specific genetic sequences from a approved options are available. And while the FDA has oversight
sample for enhanced detection and quantification. Regulations applied to on diagnostics intended for animals, it does not require 510(k)
diagnostic NAATs depend on the specific application and geographical clearance and instead places the responsibility on the manufacturer
location of use. In the United States, in vitro human diagnostics are classified to ensure the safety and efficacy of such tests. NAATs for human
as medical devices and must be authorized for use by the Food and Drug diagnostics range in complexity, with many requiring specialized
Administration (FDA) through De Novo classification or 510(k) clearance laboratories, equipment, and trained personnel, while others can be
to ensure their safety and efficacy. The FDA can also grant tests Emergency administered at the POC, including individual at-home use.

Figure 1. Point-of-care site testing offers significant time savings over centralized laboratory site testing

Point-of-care (POC) site testing

POC sites
POC
test
TIME SAVINGS
with POC site testing
NEED FOR COLLECTION ANALYSIS TEST ACTION
TESTING OF SAMPLE OF SAMPLE RESULTS PLAN

Standard centralized laboratory site testing

Standard
TRANSPORTATION test
OF SAMPLE

Testing lab PREPARATION ANALYSIS TEST REPORTING ACTION

OF SAMPLE OF SAMPLE RESULTS OF RESULTS PLAN

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Assay performance for diagnostics
Amplification of nucleic acids can be accomplished using polymerase sequences, an issue addressed through good primer design, as discussed
chain reaction (PCR) or isothermal amplification, which utilizes a later. Also, suboptimal assay conditions causing non-specific annealing
single reaction temperature to amplify the genetic target(s) of interest. or amplification can be due to the reaction formulation or excessive
Regardless of the amplification method, a diagnostic NAAT must be carryover of sample materials.
sensitive, specific and robust. These performance characteristics can
be impacted by assay parameters such as specimen type, DNA/RNA Robustness encompasses the repeatability and reproducibility of an
extraction, primer design, amplification workflow and reagent selection. assay, particularly when minor variations in test conditions occur, such as
changes in set up time, incubation temperature, reagent lots, or operator.
The measure of sensitivity refers to the true positive rate, indicating the Establishing the precision of the assay and the variation between
probability that a test correctly identifies a positive sample as positive. experimental runs is critical to ensure the assay outcomes are reliable.
This rate is affected significantly by the limit of detection (LoD),
often defined as the lowest concentration of DNA or RNA required to
achieve a 95% detection rate for a given sample type from isolation to
detection. Sources of variability must be considered when determining
the LoD, and data must be collected for multiple replicates at this
concentration to prove the analytical sensitivity of the assay. Sensitivity
may be reduced by inefficient or incomplete nucleic acid extraction,
inhibitory components in samples, or other factors that could lead to the
amplification reaction not occurring as intended.

The measure of assay specificity is the true negative rate, or the

probability that a test correctly identifies a negative sample as negative.
It is essential for the assay to precisely detect its intended target without
detecting unrelated targets. Specificity issues can arise from similar target

MDx Assay Development

Assay development with PCR by the 5´ flap endonuclease activity of Taq DNA polymerase during
PCR is currently the gold standard for nucleic acid amplification. amplification. Hydrolysis probes add an additional layer of specificity to
New assays can be designed quickly to detect nucleic acid with high the reaction, as off-target amplification products do not result in probe
sensitivity and specificity, especially given the wealth of knowledge that cleavage and are, therefore, not detected. This specificity level contrasts
has accumulated on PCR since its invention in 1983. PCR relies on the with intercalating dye-based methods that generate a fluorescent signal
thermal denaturation of DNA to facilitate strand separation, followed by for all double-stranded DNA produced and can require a melt curve post-
primer annealing and extension by a thermostable DNA polymerase, such amplification to assess specificity.
as OneTaq® Hot Start DNA Polymerase (NEB #M0481).

The approaches utilized in diagnostic PCR assays includes end-point, real-

time or digital PCR. End-point PCR assays can determine the presence
or absence of a target sequence, while real-time and digital PCR support
quantification of the target. Real-time PCR is frequently used for NAATs as
it simultaneously amplifies and detects in a single reaction vessel. Detection
is typically achieved using one or more fluorescently-labeled probes
that hybridize to the target sequence. In particular, hydrolysis probes
(e.g., TaqMan® probes) anneal to the target sequence and are cleaved

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Assay development with LAMP Strand displacement activity, an inherent property of some DNA
Isothermal amplification has emerged as an alternative to PCR, offering polymerases, is a primary feature that enables LAMP, as well as other
rapid, low-cost, molecular diagnostics with thermocycler-free convenience. isothermal amplification techniques. In contrast to PCR, strand
Before the COVID-19 pandemic, isothermal techniques like Transcription separation in LAMP is produced by the strand displacement properties
Mediated Amplification (TMA), Strand Displacement Amplification (SDA) of the large fragment of Bst DNA Polymerase (NEB #M0275) or
and Nicking Enzyme Amplification Reaction (NEAR) had been used in its engineered derivatives, Bst 2.0 (NEB #M0537), and Bst 3.0
authorized platforms. The ability to use a single temperature is attractive for (NEB #M0374).
its simplicity in device design, and as the promise of point-of-care testing
grows, isothermal techniques have been attracting more attention. When designing assay primers, we recommend designing multiple
primer sets to be tested empirically for speed, sensitivity and specificity.
Loop-mediated isothermal amplification (LAMP) uses looping primers While not common, LAMP primers can display non-template
and relies on strand displacement instead of thermal cycling (Figure 2). amplification. A candidate set should be screened with non-template
This technique has been widely studied and numerous proof-of-principle control (NTC) reactions to ensure this doesn’t occur. If the primers are
laboratory tests have been published showcasing it as an in-the-field designed with insufficient space to include both loop primers, this can
platform. LAMP was used for the first time in an FDA-authorized test be rectified by modifying the design parameters. It is important to note
in 2012. As it became clear that the SARS-CoV-2 pandemic would that incorporating even a single loop primer can substantially improve
overwhelm clinical diagnostic capacity worldwide, many developers turned reaction speed.
to LAMP for supplemental testing support and increased access to near-
patient and decentralized testing options. This increased attention resulted Recommended products for
in approved SARS-CoV-2 LAMP assays and the first-ever authorized development of PCR & LAMP assays
at-home molecular diagnostic test. Before the pandemic, LAMP was often
viewed as a challenging method for developing assays because of its • WarmStart® Fluorescent LAMP/RT-LAMP Kit
perceived complexity, lower specificity and sensitivity compared to PCR. (with UDG) (NEB# E1708)
However, through the availability of robust, high-performance reagents,
• WarmStart Colorimetric LAMP 2X Master
along with primer design tools and extensive knowledge to support assay
Mix with UDG (NEB# M1804)
development, LAMP is now recognized as a validated amplification option
for molecular diagnostics. • Luna® Probe One-Step RT-qPCR 4X Mix
with UDG (NEB# M3019)

Figure 2. Overview of LAMP workflow

3´ F3c F2c F1c B1 B2 B3 5´

5´ F3 F2 3´
c Forward internal
F1
5´ primer (FIP)

5´ F1c F2 F1 B1c B2c B3c 3´

3´ B2 B3 5´

Backward internal
B
1c

primer (BIP) 5´

3´ F1 F2c F1c B1 B2 B1c 5´

Loop B Loop F
3´ 3´
F1c B1 F1 B1c
B2c
F2c
F2

B2

B2
F2

F1 3´ 5´ B1c F1c 5´ 3´ B1 BIP

FIP
B
F1

1c
c

5´ 5´

Exponential Amplification

Loop-mediated isothermal amplification (LAMP) uses 4-6 primers recognizing 6-8 distinct regions of target DNA. A strand-displacing DNA polymerase initiates synthesis and 2 of the
primers form loop structures to facilitate subsequent rounds of amplification.
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Sample prep optimization
Molecular diagnostic amplification assays interrogate DNA or RNA Direct sample analysis with minimal sample proocessing enables
from various sample types. Commonly investigated sample types faster results and reduced diagnostics costs. Methods allowing for the
include blood, saliva, fresh/frozen tissue, FFPE tissue blocks, and interrogation of crude samples were utilized during the pandemic. One
swab samples. Each sample type presents challenges to processing and example of a successful protocol involved adding Proteinase K directly
nucleic acid extraction, which can impact downstream amplification to saliva samples, followed by a short heating step that inactivated
assays. Extraction protocols must be investigated and optimized for the virus. These saliva samples were then analyzed using RT-qPCR or
each sample type to obtain nucleic acids with minimal contaminants LAMP [4]. Many versions of simple lysis buffers based on the HUDSON
and inhibitors. composition of TCEP and EDTA proved compatible with LAMP using
various sample types [5,6,7]. Additionally, because of its robustness,
Workflows utilizing NEB’s Monarch® nucleic acid purification kits LAMP is more tolerant to the presence of inhibitors in a sample,
have been optimized to save time, decrease hands-on steps, and results allowing for crude samples to be used directly. While the best results are
in clean DNA or RNA that can be confidently transitioned into the obtained using nucleic acids purified from a sample, reliable results can
next workflow steps. Many of these extraction methods are compatible be obtained from direct analysis of samples such as saliva or samples in
with high-throughput, automated sample processing. transport media.

BLOOD SALIVA FRESH/FROZEN FFPE SWABS

TISSUE TISSUE BLOCKS

Primer design for sensitive

and specific assays
One of the most critical steps for the successful development of a The quality of the primers is also an important consideration.
molecular diagnostic assay that is frequently taken for granted is primer Standard desalting is generally sufficient for assay development,
design. Primers are complementary to the desired target sequence and but HPLC purification is beneficial for some assay deployment,
support exponential amplification of the product. However, subtle as it minimizes the presence of manufacturing byproducts, such
changes in the design can greatly impact assay performance. as truncated oligos, that can adversely impact the robustness of
the assay. This is even more critical for workflows such as LAMP,
It is important to screen several primer sets to achieve the desired as 4 to 6 primers are required for amplification of a single target
sensitivity and specificity when designing an assay. Ideally, primers rather than the traditional 2 primers for PCR. In some cases,
are created with a GC content between 40 and 60% while avoiding primers must be able to detect genetic variants of the target
hairpin loops in their secondary structure. Complementarity with other sequence (e.g., Influenza) and so degenerate primers that contain
primers in the assay should be minimized, as overlap can result in primer different bases at the variable position(s) must be included.
hybridization and off-target amplification, decreasing overall assay
performance. Given that more primers are involved in multiplex assays,
greater caution is warranted to avoid primer dimers.

6
Primer design recommendations for PCR or LAMP • LAMP: This technique utilizes a unique primer design, which
facilitates strand displacement activity of the polymerase, which is
• PCR: To assist with PCR primer design, we recommend using a critical for LAMP. The “Loop” in the LAMP name is a reference to the
program such as Primer 3. Primer pairs should be designed so that dumbbell structure formed through the core LAMP primers FIP and
their melting temperatures (Tm) are within 5°C of each other and BIP, which contain 5´ halves of downstream inverted complement
are between 50-72°C. Reaction buffer components can impact the sequence to create loops. These loops are inherently priming,
melting temperature of primers so using a Tm Calculator, like the meaning that once the reaction initiates through thermodynamic
one available from NEB, is recommended to help with this analysis. strand invasion, the combination of self-priming design and strand
A recommended starting point for the concentration of each primer displacement activity drives the reaction with no need for thermal
is 0.05-1.0 µM, but optimization is often necessary. Importantly, cycling. In total, six primers are used in a LAMP reaction: the core,
primer concentrations higher than 1.0 µM can lead to off target looping FIP and BIP; the exterior F3 and B3 to displace the initiation
amplification products, while those lower than 0.05 µM can impact product to start the reaction; and the supplemental LoopF and LoopB
reaction efficiency. PCR primers are typically 18-30 nucleotides in that bind and initiate on the ssDNA loop regions to accelerate the
length. While designing primers longer than 30 nucleotides can be reaction by providing additional initiation sites (see Figure 2, page 5).
beneficial for specificity, there is typically a trade-off in hybridization This number of primers and their design can seem daunting, but
kinetics that may reduce reaction yields. designing a full set for any given assay is quite straightforward when
facilitated by tools like NEB’s LAMP Primer Design Web Tool.
While not common, it is possible for LAMP primers to display
nontemplate amplification and a candidate set should be screened with
Figure 3. Comparison of reaction parameters of non-template control (NTC) reactions to ensure this doesn’t occur. In
LAMP and PCR assay development the design, it is possible for insufficient space to accommodate both
Loop primers; this can be adjusted in the settings, or if just one Loop
LAMP vs. PCR primer can fit it will still provide a significant enhancement to the
Temperature reaction speed. The FIP and BIP primers are quite long (generally
65˚C 45-55 bases) and are the primary drivers of the LAMP amplification.
only Varies Accordingly, their quality can have an impact on assay performance.
(cycling)
For maximum sensitivity and efficiency it is recommended to use
HPLC-purified FIP and BIP oligos when using larger scale oligo
Time
synthesis and these criteria are desirable.

20–40 min. 1.5+ hr.

Primers F Q

4–6 primers 2 primers + 1 probe

Detection Fluorescence Fluorescence

Real-time or endpoint Real-time or endpoint
Color
Turbidity
Lateral flow

Amplicon

Concatemeric Discrete

7
Assay probe designs for instrument
compatibility or lateral-flow detection
There are numerous probe-based strategies available for real-time • LAMP: While the hydrolysis probe approach is not compatible with
detection for both PCR and LAMP [8]. These probes are typically LAMP (the polymerase used has 5´→ 3´ strand displacement rather
designed with a fluorophore and corresponding quencher that can be than exonuclease activity), there are probe-based detection strategies
physically separated as a result of template amplification. The choice that can be used in LAMP. These methods fall into one of two
of the fluorophore and quencher pair will depend on the specific categories. The first uses a duplex of fluorophore-labeled primer
instrument used in the assay, as dye compatibility differs between and corresponding quenching oligo, via a Forward Inner Primer
instrument manufacturers due to differences in channel and optical filter (FIP) and backward inner primer (BIP) in an approach termed
parameters. Pairing fluorophores with high fluorescent signal intensities DARQ [9], or the Loop primer in the QUASR [10] approach. As
(e.g., FAM) with lower abundance targets may also be beneficial. A LAMP proceeds, strand displacement separates the two oligos.
guanosine should generally be avoided at the 5´ end of the probe This separation leads to the emission of fluorescence, with each
design, as it quenches the fluorescence of many reporter dyes. primer set – and consequently each amplicon – being identified by
a specific fluorophore. The second probe-based detection strategy
• RT-PCR: Hydrolysis probes are frequently used in real-time PCR uses probe binding via hybridization and separation of a dual-
(i.e., TaqMan probes). These probes are complementary to the label fluorophore and quencher. A traditional molecular beacon
target sequence and are typically 15 – 30 bases in length. It is design works in many cases but can be challenging to design.
recommended that the Tm of the probe be approximately 10 degrees Using a shorter, TaqMan - like probe will give sufficient signal
higher than the Tm of the primers. This helps ensure the probe from hybridization alone when an internal quencher is omitted.
anneals to the target sequence prior to the extension of the primers. These hybridization probes can be enhanced with Locked Nucleic
Typically, probe concentrations in PCR range from 50 – 300 nM. Acid (LNA) oligos to provide strict annealing properties and can be
designed for single-base discrimination and genotyping [11].
Figure 4. LAMP primer design for lateral flow tests
Where desired, these labeling strategies can alternatively be used for
FAM lateral flow detection, either by end-labeling primers on opposite
3´ 5´ 5´ 3´
3´ 3´
strands of the target (e.g., FIP and LoopF, FIP and BIP, etc.) or
3´ 5´ + 5´ 3´ by use of a dual-labeled hybridization probe as dictated by the
Biotin
detection moieties of the lateral flow strip.
5´ 5´

Biotin LAMP

Amplification products

Lateral flow α-FAM

antibody
– +

C
Biotin
T Control
Streptavidin
antibody
Flow

T (test) C (control)

8
Individual components versus pre-optimized
reaction formulations
Once the desired assay chemistry is selected and primers are evaluated,
choosing the ideal format for the test is important for final assembly of
the assay. In many cases, the simplest approach is an off-the-shelf master
mix (such as those included in NEB’s Luna or WarmStart LAMP product
portfolios). When bespoke individual reaction components are desired,
enzymes and buffers can be formulated specifically for a particular assay.
NEB offers optimized and flexible DNA polymerases that can be used
in the development of diagnostics. NEB also offers reverse transcriptases
for those working with RNA, including LunaScript® RT Super Mix
(NEB #M3010), WarmStart RTx Reverse Transcriptase (NEB #M0380)
and WarmStart RTx Reverse Transcriptase (Glycerol-free, NEB #M0439).
Buffer components and deoxynucleotide triphosphates (dNTPs) and NTPs
are also availble for formulation optimization To learn more about our diverse
amplification portfolio, visit:
When deciding whether to integrate a pre-optimized master mix or
[Link]/MDx
individual reaction components, consider the advantages and disadvantages
in relation to your development strategy and timeline (Figure 5).

Figure 5. Comparison of pre-optimized off-the-shelf master mixes to those with individualized components

OFF-THE-SHELF MASTER MIX INDIVIDUAL ASSAY COMPONENTS

[Pre-optimized] [Optimization Required]

Potential advantages Potential advantages

PRE-OPTIMIZED FLEXIBILITY
to work with most targets (e.g., concentration)

Potential disadvantages

REQUIRES OPTIMIZATION REQUIRES PURCHASE

of each component of individual components

EASE OF PURCHASE AUTOMATION TRACKING DYE

One mix, one vendor Friendly and ready Formulated with visible dye

Potential disadvantages

MAY NOT BE OPTIMIZED SET FORMULATION MAY REQUIRE PURCHASE

for specific target(s) (“locked-in”) from multiple vendors

9
Multiplexing amplification-based
diagnostics – advantages and disadvantages
Multiplexing permits the simultaneous detection of several targets from combining targets to maintain accuracy, sensitivity and precision.
the same sample in a single reaction [12]. A multiplexed diagnostic For optimal performance in a single reaction vessel, all assay
assay is advantageous when distinguishing between multiple causative components must coexist and not inhibit one another.
pathogens that present with similar clinical symptoms (e.g., respiratory
symptoms associated with COVID-19 and Influenza). Considerations for developing a multiplex assay are similar
to singleplex assay development, and includes factors such as
The benefits of multiplexed assays are numerous. Patient samples, primer and probe design. Additional challenges in multiplex
especially those that are challenging to collect, are precious; often, there design include one target dominating the amplification reaction,
is a limited amount of material to test. Therefore, interrogating a sample negatively impacting the assay performance for other targets, and
to obtain multiple answers simultaneously is ideal. Additionally, labs the inability to detect co-infections when one target is significantly
will see a reduction in assay setup and run time. This ultimately impacts more abundant than another. Optimization will require identifying
turn-around time and enables clinicians to share results with patients the ideal primer and probe sequences for all assay targets and their
and make informed health decisions sooner. Co-infections are also more corresponding concentrations to ensure robust amplification. The
easily identified. A reduction in consumables, reagents, and setup time additional limitations regarding instrumentation discussed earlier
will also lead to cost savings. also apply.

Despite the numerous advantages offered by multiplex assays, their

development presents a challenging undertaking. Each assay target
must be individually optimized. Further optimization is required when

10
Risk mitigation for
reliable assay results
Despite rigorous optimization and design of your assay and workflow, Historically, E. coli UDG was recommended for many diagnostic
attaining 100% accuracy is challenging. Incorporating robust and reliable NAATs. However, this E. coli enzyme is difficult to heat inactivate,
positive and negative controls in each assay will improve confidence, and residual UDG activity can degrade DNA products during
yet scenarios leading to false positives and negatives may still occur. the intended amplification, particularly in LAMP and isothermal
Implementing specific measures during assay development is critical to applications without high-temperature denaturation. E. coli UDG use
mitigating these issues. was also limited to DNA-based workflows, as its enzymatic activity
during reverse transcription is detrimental to cDNA generation.
Optimized extraction protocols that remove inhibitory compounds from New thermolabile variants, including Antarctic Thermolabile UDG
nucleic acid samples are important for reducing the risk of false negatives. (NEB #M0372), support isothermal and RNA-based workflows
Ensuring the assay has a wide dynamic range and appropriate LoD for a and are rapidly heat inactivated at temperatures typically used for
gene target using a given primer and probe design is also a critical measure reverse transcription (e.g., 50°C). Many of NEB’s qPCR, RT-qPCR,
of assay sensitivity. and LAMP/RT-LAMP reagents include dUTP and/or Antarctic
Thermolabile UDG in pre-mixed and optimized formulations for
False positive results can often be traced to contamination and laboratory
carryover contamination prevention.
error. Contamination can include cross-contamination from other samples,
DNA residue contamination on instruments and surfaces, or carryover
contamination from previously amplified PCR reactions. The most common
Figure 6. Evaluation of RT-qPCR carryover prevention
source of cross-contamination comes from amplicons becoming aerosolized
following amplification, particularly when the reaction vessel is opened
for analysis. To reduce false positives via cross-contamination, consider
integrating these steps into your lab protocols: Carryover contamination
from previous RT-qPCR (with dUTP)

• Ensure your assay proceeds in a unidirectional manner in the U U

laboratory, moving from pre-PCR to post-PCR.
U
No UDG UDG
• Isolate initial sample processing and reaction setup from
instrumentation for amplification in separate designated spaces U U Y Y
U Y
• Ideally, combine amplification and analysis into a single step that
Subsequent Subsequent
does not require opening the reaction vessels (e.g., real-time PCR) RT-qPCR RT-qPCR

• Designate a third space for analysis if reaction tubes will be opened

False Positive True Negative
post amplification
RFU

RFU

Amplified
In real-time PCR and LAMP methodologies, amplification and analysis
are performed in a closed vessel, reducing the risk of amplicons becoming Not amplified
aerosolized. Integrating dUTP and Uracil-DNA Glycosylase (UDG) into the Cycles Cycles
amplification protocol adds an additional layer of carryover contamination U = uracil Y = abasic site
risk mitigation. Including dUTP into the amplification mix ensures that
synthesized amplicons contain uracil due to the efficient incorporation by To evaluate the capacity of carryover product digestion, a uracil-containing RT-qPCR product was
DNA polymerases; Taq and Bst 2.0”. These uracil-containing templates generated using the Luna One-Step Probe RT-qPCR kit. Approximately 105 copies of the uracil-
containing product were used as template for subsequent qPCR reactions using three different
can then be recognized by UDG during an initial step in the amplification RT-qPCR master mixes (all containing UDG). Following manufacturer’s recommended protocols,
workflow. UDG excises uracil, creating an abasic site, preventing the incubation at 25˚C at the start of the 2nd reaction enables UDG to degrade the dU-containing input,
polymerase from amplifying the contaminating DNA sequences. Building reducing its ability to contribute to a (false) positive signal. The ΔCq value is the cycle difference
between carryover treatment and no carryover treatment of the same input. Larger ΔCq values
this carryover prevention into the assay can significantly reduce the risk of indicate more efficient carryover product digestion. After decontamination using the Luna mix, full
false positives. product digestion was observed in one sample and a large ΔCq was observed in the second. Note
that the amount of DNA present in true contamination events is difficult to assess/predict.

11
Considerations for developing
a lyophilized molecular
diagnostic assay
The COVID-19 pandemic highlighted the value of molecular diagnostic assays that could be
performed at home or in point-of-care settings. There is also increasing interest in field-deployable
diagnostics in agriculture to assess crop pathogens on-site so that appropriate disease management
can be enacted quickly. While wet reagents are an acceptable solution for testing in a centralized
lab, eliminating the need for cold transport and storage is a key factor in enabling testing in non-
laboratory settings or in people’s homes. Lyophilization, or freeze-drying, offers a robust solution
for ambient room-temperature shipping and storage with several additional benefits, which are
highlighted below [13] (Figure 7).

Figure 7. Benefits of lyophilized products

REMOVAL OF COLD MINIMIZED ASSAY MAXIMIZED STABILITY

CHAIN REQUIREMENT CONTAMINATION Formulation optimized
Reduced logistical burden on Due to reduced for stability
order fulfillment and storage pipetting steps

Benefits of
Lyophilized Products

MORE EFFICIENT LONGER REDUCED ENERGY

ASSAY SETUP SHELF LIFE CONSUMPTION
Due to automation Ambient temperature Throughout product
storage shelf life

12
Creating a high-performing and robust lyophilized assay requires additional design and formulation considerations. Deciding early in
the development phase whether to have an assay in a lyophilized form will save time and effort over the long term, reducing the need for
potential re-formulations, re-design, and re-optimization of kit reagents.

Transitioning to a room temperature stable, lyophilized dry assay is not as simple as freeze-drying, wet reagents. Often, it requires the wet
reagents to be re-formulated, with the following parameters being taken into consideration [14]:

• Master Mix Composition: Common master mix components, such Lyophilizing an assay is a multi-faceted development process, but
as glycerol and co-solvents, are either incompatible with lyophilization it offers many advantages over wet reagents. Typically, building
or adversely affect the reagent’s stability. Some of these components an assay based on commercially available products like LyoPrime
may help with enzyme stabilization and assay robustness. Should the Luna® Probe One-Step RT-qPCR Mix with UDG (NEB# L4001)
exclusion of these chemicals negatively impact performance, alternative and LyoPrime WarmStart™ Fluorescent LAMP/RT-LAMP Mix (with
components may be used to restore the performance of the mix, which UDG) (NEB #L4401) can be more straightforward and cost effective.
can be evaluated through substitution testing. However, it is possible to build a lyophilized assay from individual
enzymes and buffer components using glycerol-free enzymes. NEB
• Excipient Selection: Excipients, also known as cryoprotectants, are provides technical guidance and support, strategic reagent selection,
essential to protect the enzymes during lyophilization and provide and tailored formatting recommendations to meet desired assay
long-term ambient temperature stability. They give the dried material specifications.
a solid, cake-like structure and allow for rapid dissolution. Excipient
selection should be carried out for each intended application to ensure
assay performance. Optimizing the excipients initially will save time and
reduce costs by preserving the reagent’s activity.

• Freeze-Drying Cycle Parameters: The lyophilization process involves

freezing a master mix at a low temperature (~ -40°C) and low pressure
(~ 100 mTorr). Optimizing parameters, such as drying temperature and
To learn more about whether a
ramp rate, allows for the creation of a dried, lyophilized cake with no lyophilized assay is right for you,
residual moisture that is easy to reconstitute. Poorly optimized freeze- visit [Link]/lyosciences
drying cycles can result in incomplete drying and reagent collapse or visit the following resources:
or over-drying, resulting in an assay with poor enzyme performance.
Investing time in designing the right drying cycle will allow for the
• Enhancing Molecular Diagnostics in Regulated Markets
ease of scalability and increased efficiency.
with Lyophilized Assays
• Packaging: The packaging design for the lyophilized product should
• Key considerations for optimal lyophilized reagent
provide protection from moisture and atmosphere, as it will begin
development
“aging” on exposure. Glass is the best primary container for protecting
lyophilized products since it is impervious to atmospheric oxygen • Debunking the myths: clearing up six common
and moisture. If plastic is chosen as the primary packaging container, misconceptions about reagent lyophilization
secondary packaging, such as a moisture barrier foil pouch, is required.
This is because plastic’s porous structure will allow atmospheric
moisture to leak into the lyophilized cake. The secondary packaging is
often sealed under an inert atmosphere, such as nitrogen gas, and may
include a desiccant to absorb residual moisture.

13
Choosing a partner focused We deliver

on enabling your science – With a long and established history in the

marketplace, NEB has been a key supplier in
now and in the future the life sciences industry since its inception
in 1974. With a robust and reliable supply
chain network, we can supply customers with
Often, the initial focus when developing a molecular diagnostic assay is on creating and validating what they need, when they need it. We provide
the assay’s performance and protocol, leaving key considerations such as scale-up, automation custom solutions, flexibility and short lead
compatibility, lyophilization, and regulatory compliance sometimes overlooked until later stages. times to product quantities from bench to
This approach can limit advancement options. Additionally, factors like manufacturing, distribution commercial-scale.
logistics, geographic scope, and supply chain accessibility are frequently deferred, potentially
The combination of 50 years of manufacturing
delaying assay implementation.
experience, scientific expertise and an
Early engagement with suppliers and reagent providers like NEB can be a critical step when you need extensive global supply network uniquely
positions NEB to provide market insights that
access to technical expertise and optimized reagents when looking to accelerate your development
meet your ever-developing process needs and
objectives. Proactive communication about project timelines, forecasts, quality specifications, and
provide business continuity.
formatting requirements with suppliers ensures the timely availability of necessary reagents in the
required format and specifications, facilitating a more efficient and streamlined development process. Visit:
NEB offers a foundation for customers developing molecular diagnostics that pairs reagents of the [Link]/
highest quality with deep insights on approaches that accelerate the development of robust assays to innovation
fulfill the promise of accessible and reliable testing. to learn more

“ The COVID-19 pandemic highlighted the power of interdisciplinary collaboration in

driving scientific breakthroughs and innovation. From the start, our SalivaDirect team at
the Yale School of Public Health relied on the quality of not only NEB reagents but their
technical guidance and willingness to support scientific innovation, in any way possible.
NEB quickly proved instrumental to our efforts and an incredible pillar of support, offering
invaluable support in areas where we did not have sufficient resources or bandwidth. This
support extended to clinical labs around the US, with NEB working quickly to test and
demonstrate the continued efficacy of the SalivaDirect PCR test for the sensitive and reliable
detection of emerging SARS-CoV-2 variants. Importantly, NEB’s support was not unique to
us. Their team was always ready to discuss how we could work together to support testing
efforts around the world, particularly in underserved communities where outbreaks were
most acute. Their support of us and others really underscored to us their commitment to

”
scientific progress and global health.

Anne Wyllie, Ph.D.

Research Scientist in Epidemiology of
Microbial Diseases, Yale School of Public Health

14
References
1 Quyen, T., et al. (2019) Front Microbiolog., DOI: 10.3389/fmicb.2019.02234
2 Zhang, Y. & Tanner, N., (2021) Biotechniques, DOI: 10.2144/btn-2020-0157
3 Zhang, Y. & Tanner, N., (2022) Biotechniques, DOI: 10.2144/btn-2022-0096
4 Vogels, C.B.F. et al., (2021) Med, DOI: 10.1016/[Link].2020.12.010
5 Anahtar, M.N., et al.,(2020) Open Forum Infect Dis., DOI: 10.1093/ofid/ofaa631
6 Li, Z., et al. (2022 ) PLoS One, DOI: 10.1371/[Link].0268692
7 Moore, K.J. et al. (2021) Biomol Tech., DOI: 10.7171/jbt.21-3203-017
8 Navarro, E. et al., (2014) Clinica Chimica Acta, DOI: 10.1016/[Link].2014.10.017
9 Tanner, N., Zhang, Y. and Evans, TC. (2012) Biotechniques, DOI: 10.2144/0000113902
10 Ball, C.S. et al., (2016) Anal Chem., DOI: 10.1021/[Link].5b04054
11 Zhang, Y. and Tanner, N.A., (2022) Biotechniques DOI: 10.2144/btn-2022-0096
12 Wittwer, C.T. et al. (2001) Methods, DOI: 10.1006/meth.2001.1265
13 Loeb, J., Tanner, N. & Lee, M., [Link] Feature Article, Enhancing Molecular Diagnostics in Regulated Markets with Lyophilized Assays
14 M.A., Chiu, S.G., Lee, D.R. & Nichols, N.M. White Paper, Key considerations for optimal lyophilized reagent development

How can NEB support your

diagnostics development efforts?
Integrating scientific expertise with a global presence, NEB can support your amplification reagent
needs for small-scale research projects through large scale manufacturing and commercialization. Setting
the standard for an effective partner, NEB provides personalized technical and commercial support
throughout a project’s entire life cycle. We are committed to your success.

Contact our Customized Solutions Team today to find out

how we can support your next MDx development project at
[Link]/customized-solutions

“ We look to our diagnostics customers as the real innovators. These customers

engage with NEB scientists to understand what our products do and don’t do,
to make good decisions quickly about whether we are a fit for their technology
platform. We also provide clarity about capacity, turn-around-times and when
possible, pass along cost savings to our customers when scale can be achieved to

”
reduce the price of our products.

Salvatore V. Russello, Ph.D.

CEO of New England Biolabs

15
Setting the standard
for an effective partner
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