Endpoint vs. Real-Time Recombinase Polymerase Amplification (RPA)

Endpoint RPA supports several detection strategies, allowing you to tailor the readout.

• Lateral Flow Assays (LFA): A gold standard for rapid, point-of-care diagnostics, lateral flow assays are most commonly used with RPA through dual-labeled amplicons that enable simple, visual detection on a test strip. This approach requires minimal equipment and delivers fast, easy-to-interpret results, making it ideal for field and low-resource settings.

Note: An emerging enhancement to this workflow leverages RPA–CRISPR synergy. In this approach, the amplified product activates a CRISPR-associated enzyme (e.g., Cas12a), which cleaves a reporter molecule via collateral (“trans”) cleavage activity. This added detection layer can improve sequence specificity and reduce background signal from non-specific amplification or primer-dimer artifacts.

• Colorimetric Detection: This method relies on a visual color change to indicate successful target amplification. By incorporating specific dyes or gold nanoparticles, you can instantly verify the presence of a target sequence.
• Electrophoresis: For laboratory settings, gel electrophoresis provides a reliable way to visualize products and confirm exact fragment sizes during the initial assay development and optimization phases.

Endpoint RPA shines in scenarios where speed, simplicity, and accessibility are paramount. It is the preferred format for diagnostic development in remote clinics, agricultural field testing, and low-resource environments, where minimal equipment and clear visual outputs are essential. By relying on straightforward readouts, endpoint RPA enables rapid pathogen detection without the need for complex instrumentation.

This format is widely used for identifying infectious agents such as MRSA and HIV, as well as for veterinary and agricultural testing, including plant pathogen detection and food safety screening for threats like Listeria monocytogenes. When combined with CRISPR-based detection, endpoint RPA can achieve enhanced specificity while maintaining its low-complexity workflow, making it a powerful and scalable solution for decentralized testing.

Explore our RPA Endpoint Kit and RT-RPA Endpoint Kit to learn more.

Real-time RPA relies on the use of target-specific detection probes—specialized oligonucleotides labeled with both a fluorophore and a quencher. During the amplification process, these probes bind to the target sequence and are enzymatically cleaved, separating the fluorophore from the quencher to emit a measurable light signal.

Note: Synthego’s Real-Time RPA kits are specifically designed to utilize Exo probes, which are optimized for high-sensitivity, real-time fluorescent detection.

Alternatively, CRISPR-Cas can be integrated into real-time workflows to simplify assay design. In CRISPR-RPA kinetic assays, detection relies on cleavage of a universal fluorescent reporter, removing the burden of designing complex, target-specific probes and leveraging gRNA-mediated targeting for specificity.

Real-time RPA is indispensable for high-throughput and time-critical diagnostic workflows, particularly in clinical reference laboratories. It provides the automation, quantitation, and contamination control required for rigorous testing environments, enabling users to monitor amplification in real time without opening the reaction vessel. This makes it ideal for quantifying viral loads, such as HIV-1 proviral DNA, and for tracking pathogen burden across a wide range of infectious diseases with rapid turnaround times.

Beyond centralized labs, real-time RPA is also well-suited for point-of-care, environmental, and food safety applications where sensitive, reproducible detection is required. Its closed-tube, fluorescence-based format minimizes contamination risk while supporting semi-quantitative analysis of bacterial, viral, and antimicrobial resistance targets. In research and assay development workflows, real-time RPA further enables rapid optimization and kinetic analysis, especially when paired with CRISPR-based detection to enhance specificity.

Explore our RPA Real-Time Kit and RT-RPA Real-Time Kit to learn more.