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| Product Name | RNA Rapid Isothermal Amplification Kit (Chromatographic, Nfo Probe) |
| Catalog No. | RESK-0117 |
| Description | A raw-material reagent kit for recombinase polymerase amplification (RPA), developed with recombinase, single-stranded DNA binding protein, and strand-displacing DNA polymerase for isothermal amplification of RNA nucleic acid targets. When paired with a customer-designed Nfo probe and a biotin-labeled reverse primer, results can be read directly on a lateral flow chromatographic strip. Building on the basic (gel-based) RPA reagent, this kit additionally contains endonuclease IV (nfo). During amplification, when the Nfo probe hybridizes to its complementary template strand, endonuclease IV recognizes and cleaves the THF residue site, generating an amplicon carrying both the probe label and the primer label; after adding the corresponding labeled antibodies, a result band appears on the test line of the strip. |
| Intended Use | For isothermal amplification and rapid detection of RNA targets in research and assay development; widely applicable to in vitro diagnostics, veterinary testing, food safety, biosafety, and agriculture. |
| Principle / Technology | Recombinase polymerase amplification (RPA): recombinase proteins form complexes with primers that search for homologous sequences on double-stranded DNA; the recombinase strand-exchange activity inserts the primer at the homologous site while single-stranded binding proteins stabilize the displaced DNA strand. The recombinase is then disassembled, leaving the primer 3' end accessible to a strand-displacing DNA polymerase that extends the primer; repeating this cycle exponentially amplifies the target region. Multiple enzymes participate, including T4 UvsX, T4 UvsY, GP32, Bsu DNA polymerase, and creatine kinase (CK). This kit additionally includes endonuclease IV (nfo), which recognizes and cleaves the THF (tetrahydrofuran) residue site of a hybridized Nfo probe to produce a dual-labeled amplicon. |
| Detection Method | Lateral flow chromatographic strip readout using the Nfo probe method. The user designs an Nfo probe and a biotin-labeled reverse primer. When the Nfo probe is complementary to the template strand, endonuclease IV cleaves the THF residue site, yielding an amplicon that carries both the probe label (e.g., FAM) and the primer label (e.g., biotin). Addition of the corresponding labeled antibodies produces a visible band on the test line of the chromatographic strip. |
| Sample Type | RNA nucleic acid templates. |
| Performance Range / Specifications | Multi-enzyme system involving T4 UvsX, T4 UvsY, GP32, Bsu DNA polymerase, and CK. Isothermal reaction at 37-42 °C for 10-30 min amplifies trace nucleic acid to a detectable level. Detection sensitivity down to a single copy or tens of copies (or fewer) of RNA. Low instrument dependence; simple operation and straightforward result interpretation. Widely used in in vitro diagnostics, veterinary, food safety, biosafety, and agriculture. |
| Sensitivity / LOD | Down to a single copy or tens of copies (or fewer) of RNA. |
| Specificity | Sequence-specific detection achieved through the combination of specific primer pairs and the Nfo probe, which is cleaved by endonuclease IV only when complementary to the template strand. |
| Reaction Conditions / Protocol | Isothermal incubation at 37-42 °C for 10-30 min. Recommended primer length is 30-35 bp, avoiding complex secondary structures; the 5' end of the downstream primer must carry a label matching the strip (e.g., biotin or digoxigenin). Recommended amplicon length is 100-500 bp. The Nfo probe is about 46-52 nucleotides long, with approximately 30-35 bp between its 5' end and the THF position and about 15 bp between the THF position and its 3' end; the 5' end carries an antigenic label (typically FAM or carboxyfluorescein), the interior contains a nucleotide-analog substitution (e.g., a tetrahydrofuran residue, THF), and the 3' end carries a polymerase extension-blocking group (e.g., C3-spacer, phosphate, or dideoxynucleotide). |
| Components / Formulation | RPA enzyme system including T4 UvsX recombinase, T4 UvsY, GP32 single-stranded DNA binding protein, Bsu DNA polymerase, and creatine kinase (CK), plus endonuclease IV (nfo) for probe cleavage; reverse transcription capability for RNA templates. Primers, Nfo probe, and template are supplied by the user. |
| Storage Conditions | Store at -25 to -15 °C. Aliquot the reagents in advance before use. |
| Shelf Life | 1 year. |
| Package Specifications | 16 T; 48 T; 100 T |
| Product Form | Liquid reagent kit (isothermal amplification raw-material premix). |
| Expiration Date / Stability | Valid for 1 year when stored at -25 to -15 °C; aliquot reagents in advance to avoid repeated freeze-thaw. |
| Compatibility | Compatible with user-designed Nfo probes and labeled reverse primers matched to the lateral flow strip in use (e.g., FAM/biotin or FAM/digoxigenin label pairs). |
| Application Notes / Precautions | The THF mark replaces a base within the probe sequence; it is not an additional inserted label. Before starting the experiment, verify that the 5' label on the probe and the 5' label on the downstream primer match the lateral flow strip being used. Avoid complex secondary structures during primer design; recommended primer length is 30-35 bp and amplicon length is 100-500 bp. Aliquot reagents in advance. |
For research use only, not for clinical use.
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