Dual-labelled hydrolysis probes typically contain a 5′ reporter dye and a 3′ quencher and generate fluorescence following polymerase-mediated probe cleavage. Other probe formats, including Molecular Beacons and Scorpions Primers, utilise hairpin structures that release fluorescence upon target-specific hybridisation rather than probe cleavage. Because fluorescence is produced only when the probe hybridises to the intended target, these assays provide highly specific detection across a wide range of applications, including SNP genotyping, pathogen detection, gene expression analysis and multiplex qPCR. Assay performance is influenced by probe design, melting temperature (Tm), hybridisation kinetics, sequence specificity, fluorophore and quencher selection, probe placement within the amplicon and overall assay optimisation.
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How probe-based qPCR works
Supporting sensitive, target-specific nucleic acid detection
Who uses probe-based qPCR assays?
Molecular biology researchers
qPCR assay developers
Molecular diagnostics teams
SNP genotyping specialists
Clinical research laboratories
Agricultural genomics laboratories
Gene expression researchers
Pathogen detection laboratories
Translational research organisations
Multiplex assay development teams
When to use probe-based
qPCR assays
Probe-based qPCR is particularly valuable when applications require sequence-specific detection, SNP discrimination, multiplex target analysis or increased confidence in target identification beyond primer-mediated amplification alone. The additional sequence-specific hybridisation event provided by the probe introduces another level of assay specificity, helping distinguish true amplification from non-specific products. Probe chemistry selection should be guided by target sequence characteristics, assay objectives and multiplex requirements.
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Choose hydrolysis probes for quantitative detection
Dual-labelled hydrolysis probes leverage polymerase-mediated probe cleavage to generate fluorescence that track target amplification, supporting quantitative PCR, gene expression analysis and routine real-time PCR workflows.
Use MGB, LNA or BHQplus chemistries for difficult targets
Targets containing A/T-rich regions, short sequences or SNPs may benefit from probe chemistries that increase duplex stability and improve mismatch discrimination.
Choose fluorophore combinations for accurate detection
Applications requiring simultaneous detection of multiple targets benefit from carefully selected fluorophore and quencher combinations that minimise spectral overlap, reduce channel cross-talk and preserve quantitative accuracy.
Applications and technologies related to probe-based assays
Applications
Probe-based qPCR assays are widely used for molecular diagnostics, pathogen detection, SNP genotyping, gene expression analysis and agricultural genomics, where sequence-specific detection and reliable target discrimination are critical to decision making.
Technologies
Explore the complementary molecular technologies used alongside probe-based assay workflows, from PCR amplification and real-time detection to dye-based qPCR and advanced sequencing approaches.
Featured Resources
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LGC expertise in probe-based assay design
and development
Supporting scientists with probe selection, assay optimisation and multiplex workflow development.
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Global reach
144
Countries served through our global diagnostics and genomics network.
Product portfolio
11,000+
Products supporting diagnostics, genomics and life science workflows.
Frequently asked questions
How do probe-based qPCR assays work?
Probe-based assays use fluorescently labelled probes that hybridise to the target sequence and generate fluorescence only when the intended target is detected, enabling real-time, sequence-specific detection and quantification. Different probe chemistries generate signal through different mechanisms, including polymerase-mediated probe cleavage (hydrolysis probes), target-induced conformational changes (Molecular Beacons) and intramolecular probe-target interactions (Scorpions™ Primers).
What is the difference between a probe and a primer?
Primers initiate DNA synthesis during PCR amplification. A qPCR probe provides an additional sequence-specific detection mechanism, generating fluorescence only when the intended target sequence is detected.
When should you use a probe instead of dye-based assays?
Probe-based assays are commonly selected when higher specificity, multiplexing capability or greater confidence in target identification is required.
What is a Black Hole Quencher® (BHQ®) and why does quencher selection matter?
Black Hole Quenchers® (BHQ®) are non-fluorescent quenchers that dissipate reporter emission as heat rather than fluorescence, helping reduce background signal and improve assay signal-to-noise ratio. Appropriate quencher selection also supports multiplex assay design by contributing to optimal fluorescence performance across detection channels.
Which probe chemistry should I choose: hydrolysis probes, Molecular Beacon Probes, or Scorpions™ Primers?
Probe selection depends on target characteristics and assay requirements. Hydrolysis probes, including dual-labelled BHQ™ Probes, are commonly used for routine quantitative PCR; MGB, LNA, and BHQplus™ Probes are often selected for SNP genotyping and short target regions, while BHQnova™ Probes are particularly suitable for A-T rich templates and targets requiring longer probe sequences.
Molecular Beacons support highly specific hybridisation-based detection, and Scorpions™ Primers can provide rapid signal generation where combined primer-probe functionality is advantageous. Both probe formats support melt curve analysis and are ideal for SNP/mismatch discrimination.
How many probes can be multiplexed in one reaction?
Multiplex capacity depends on instrument optics, fluorophore selection, spectral overlap and assay optimisation. Practical multiplexing limits are often determined by the number of available detection channels and their ability to resolve fluorescence signals. Probe and primer interactions and overall assay performance should also be considered during multiplex assay design.
How do you design a qPCR probe?
Important considerations include probe Tm relative to primer Tm, amplicon length, target specificity, probe placement between primer binding sites, SNP or mismatch positioning (if applicable), fluorophore and quencher selection, and avoidance of secondary structures that could impair hybridisation or signal generation.
What drives the cost of a custom probe?
Probe chemistry, fluorophore and quencher selection, purification requirements, probe length and specialised modifications can all influence probe manufacturing complexity and overall cost.
What purification and QC do your probes come with?
Purification and QC specifications vary according to probe chemistry, fluorophore selection and manufacturing requirements. Refer to the relevant product documentation for detailed purification and quality control information.
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Need help designing a
probe-based qPCR assay?
Whether you are selecting a probe chemistry, designing a multiplex panel or optimising fluorophore and quencher combinations, our scientific team can help identify the most suitable assay approach for your application.