RPA

RPA is a DNA amplification technique designed for rapid, sensitive, and specific nucleic acid detection under isothermal conditions (similar to LAMP), avoiding the need for thermal cycling. Initially invented by Piepenburg et al., 2006, its distinguishing features are:

Principle of Amplification:

• RPA uses a recombinase-primer complex to facilitate homologous pairing of primers with complementary target DNA sequences.
• The recombinase binds primers and scans the target DNA for homologous sequences, enabling strand exchange and primer binding.

Mechanism:

• Recombinase Proteins: Form a filament with primers to guide them to their complementary sequences on the target DNA.
• Single-Strand Binding (SSB) Proteins: Stabilize the displaced single strand during primer binding.
• DNA Polymerase: Extends the primers and displaces the downstream strand, enabling continuous amplification at a constant temperature (37–42°C).

Products:

• RPA generates amplified DNA fragments in a sequence-specific manner. Amplification is exponential and can be detected in real-time or endpoint assays using fluorescent probes, lateral flow strips, or gel electrophoresis (refer to the original paper).

Advantages:

• Speed: Amplification occurs rapidly, typically within 20–40 minutes.
• Low Temperature: Operates at a constant temperature close to physiological conditions (37–42°C).
• High Sensitivity: Detects low amounts of DNA, often in the range of femtograms.
• Versatility: Works well with crude samples and minimal preprocessing.
• Field Deployable: Requires minimal equipment, making it suitable for point-of-care or field-based applications.

Applications:

Initially developed for DNA detection, RPA has since been applied to various fields, including infectious disease diagnostics, environmental monitoring, and food safety testing.

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