ATTO™ dyes for double dye probes: consistent performance in qPCR and dPCR assays
This article reviews the key characteristics of high-quality fluorophores and examines how ATTO™ dyes perform in multiplex PCR applications compared with commonly used fluorescent dyes.
In probe-based qPCR and digital PCR (dPCR), fluorophores are critical components that directly influence signal detection, assay sensitivity, and multiplexing performance. The choice of fluorescent dye affects not only analytical performance but also assay design flexibility and commercialization opportunities.
Among the fluorophores available for molecular diagnostics and research applications, ATTO™ dyes have gained attention for their combination of strong fluorescence properties and broad spectral coverage. In addition, Eurogentec provides these dyes within an IP-free framework, offering greater flexibility upon assay commercialization.
1. What defines a high-quality fluorophore for qPCR and dPCR?
Selecting fluorescent reporter dyes involve more than matching excitation and emission to an instrument channel. Several characteristics influence assay performance and long-term reliability:
- High brightness
- Strong photostability
- Thermal stability
- Distinct spectral properties
- Thermal stability
- Instrument compatibility
Bright fluorophores generate stronger fluorescence signals, facilitating target detection and improving assay sensitivity, particularly when target concentrations are low.
Repeated excitation during PCR cycling can lead to photobleaching. Photostable dyes maintain their fluorescence over time, preserving signal quality throughout the experiment.
PCR assays expose fluorophores to repeated heating and cooling cycles. Thermally stable dyes maintain their optical properties during amplification.
For multiplex assays, fluorophores should have clearly separated excitation and emission spectra to minimize channel overlap.
PCR assays expose fluorophores to repeated heating and cooling cycles. Thermally stable dyes maintain their optical properties during amplification.
A suitable fluorophore should be compatible with standard optical filters and detection systems commonly used in qPCR and dPCR platforms. These characteristics collectively determine how effectively a fluorophore supports reliable target detection and multiplex assay design.
2. Why choose ATTO™ fluorophores?
Broad spectral coverage and robust fluorescence properties
ATTO™ dyes combine several characteristics that are particularly valuable for Probe-based PCR applications. They are known for:
- High fluorescence quantum yields
- Strong absorption coefficients
- Excellent photostability
- High thermal stability
- Consistent signal generation across amplification runs
- Coverage of the visible and near-infrared spectrum
The robust fluorescence properties of ATTO™ dyes are linked to their rigid chromophore structure, which contributes to both photostability and thermal stability. This structural rigidity helps limit non-radiative energy loss and reduces degradation under demanding experimental conditions.
This approach helped standardize production, reduce hands-on time and accelerate LDT validation steps.
Measurable benefits in routine Legionella diagnostics
Switching to Eurogentec’s pre-filled strip format delivered immediate and tangible benefits.
According to Dr. Laetitia Béraud, Biologist in Legionella National Reference Center, the simplified workflow had a direct impact on daily laboratory operations. She first highlighted the ease of use of pre-filled strips:
“The main advantage is the technical easiness.”
By eliminating daily reagent preparation and hands-on mixing, the new format allowed operators to focus on higher-value tasks that are essential in a reference laboratory environment. As Dr. Béraud explained, workflow efficiency is critical for routine diagnostics:
“Routine screening has to be fast and cause no technical issues. The pre-filled strips are easy to use and let our operators spend more time on typing or epidemiological techniques, which are more demanding.”
She also emphasized how documentation and traceability, often complex with fully in-house assays, became significantly easier to manage. Instead of handling primers and probes as separate components replaced at different times, all reagents are now linked to a single strip lot:
“We now change all reagents together with each strip lot. That greatly simplifies reagent follow-up and traceability.”
Standardized cleanroom manufacturing and robotic dispensing further strengthened assay robustness, notably by addressing fluorescence variability between wells, sometimes observed with in-house homogenization (see Figure 1). Dr Béraud noted:
“In-house homogenization and distribution can lead to initial fluorescence variability. With pre-filled strips, this issue is resolved.”
Figure 1. Robotic dispensing of premixed primers and probes eliminates well-to-well fluorescence variability, improving assay consistency.
Finally, she underlined the importance of responsive technical support throughout assay setup and optimization:
“You provided valuable guidance, notably in selecting the probes. We relied on your expertise, which helped us save time,” she added, highlighting how technical expert support was especially valuable and helped her save time.
From commercial kit discontinuation to long-term LDT optimization
What began as a kit supply disruption ultimately became an opportunity for the Legionella NRC to streamline its diagnostic workflow, strengthen regulatory readiness, and build a scalable custom Legionella testing solution.
Next steps
Encouraged by the success of this first outsourced assay preparation project, the NRC is now extending the approach to a second primer/probe premix. This second assay will be dispensed into laser-marked strips to facilitate differentiation between the two assays while maintaining the standardized manufacturing, traceability, and consistency required for routine diagnostic use.
Supporting LDT production with confidence
This case study illustrates how Eurogentec supports diagnostic laboratories in securing testing continuity, simplifying LDT preparation and validation, in line with IVDR requirements. Without compromising flexibility or assay design control.
Your LDT. Your design. Our preparation expertise.
Whether you need to secure diagnostic continuity or simplify routine assay preparation, we manufacture and dispense your custom qPCR premixes according to your specifications.
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