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| Product Name | Reverse Transcriptase (RT) Activity Detection Kit |
| Catalog No. | ETR-HMM-0071 |
| Description | A sensitive non-radioactive reverse transcriptase activity assay based on a PCR-enhanced detection system. Reverse transcriptase present in a sample synthesizes complementary DNA from a provided RNA template, and the cDNA product is subsequently detected and quantified by real-time PCR or colorimetric assay to provide a highly sensitive measure of retroviral or recombinant RT enzyme activity. |
| Intended Use | Detection and quantification of reverse transcriptase enzyme activity in retroviral preparations, recombinant RT enzyme quality control, biosafety testing of biopharmaceutical products for retroviral contamination, and research into RT inhibitor efficacy for antiretroviral drug development. |
| Principle / Technology | The PERT (Product-Enhanced Reverse Transcriptase) assay principle: endogenous or recombinant reverse transcriptase copies an exogenous poly(A)/oligo(dT) RNA:DNA template:primer hybrid to generate poly(dA) cDNA; the cDNA product (or alternatively, a specific RNA template transcript) is then detected by colorimetric ELISA-based hybridization or quantitative real-time PCR using a labeled probe; the signal intensity is proportional to cDNA accumulated by RT activity. |
| Detection Method | Colorimetric ELISA-based hybridization at 450 nm for the nucleic acid detection component, or fluorescence (Ct values from qPCR) for ultra-sensitive PCR-PERT format; also available as a simple colorimetric microplate format |
| Sample Type | Viral particles and viral lysates from retrovirus-producing cell cultures (HIV, MLV, MMTV supernatants), purified recombinant reverse transcriptase (HIV-1 RT, M-MLV RT, AMV RT), biopharmaceutical cell culture supernatants tested for retroviral safety |
| Performance Range / Specifications | Detection sensitivity: 10^-5 mU/mL RT activity in purified enzyme preparations; able to detect retroviral RT activity from approximately 10–100 HIV particles per mL using qPCR-PERT format; ELISA-based format detects 10^-3 mU/mL |
| Sensitivity / LOD | Lower detection limit of approximately 10^-5 mU/mL RT using the PCR-PERT format, enabling detection in a single virion produced per mL by productively infected cell cultures |
| Specificity | The poly(A)/oligo(dT) template:primer system is specifically copied by RNA-dependent DNA polymerases (reverse transcriptases) and is not appreciably copied by cellular DNA polymerases or RNA polymerases under the assay conditions; DNase I pre-treatment of samples eliminates background from residual DNA template; AZT inclusion demonstrates RT-specific inhibition for specificity verification |
| Reaction Conditions / Protocol | Lyse virus or cell pellet in lysis buffer; remove cellular debris by centrifugation; add 5 μL lysate to RT Reaction Buffer containing poly(A)/oligo(dT) template:primer hybrid; incubate 60–90 minutes at 37°C; for ELISA format: hybridize cDNA product to capture probe on microplate, detect with anti-DIG-AP antibody and pNPP substrate; for PCR-PERT format: amplify cDNA aliquot by qPCR using provided TaqMan primers; calculate RT activity from reference curve |
| Components / Formulation | Template-Primer Hybrid (poly(A)/oligo(dT), 25 nM), RT Reaction Buffer (Tris-HCl, MgCl2, KCl, DTT, dTTP), Lysis Buffer, Hybridization Buffer, Anti-DIG-Alkaline Phosphatase Conjugate, pNPP or ABTS Substrate, Wash Buffer, Positive Control RT (recombinant AMV RT with certified activity), AZT inhibitor for specificity control; for PCR-PERT variant: TaqMan primer-probe set for qPCR detection |
| Storage Conditions | Template-primer hybrid: -80°C for 12 months; RT Reaction Buffer: -20°C for 12 months; hybridization and detection components at 2–8°C; all components protect from nuclease contamination |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 96-well ELISA format (80 samples), or PCR-PERT format with qPCR compatibility |
| Product Form | Concentrated liquid and lyophilized reagent components with certified positive control RT |
| Quality Control | Each lot tested with certified AMV RT positive control across 4–5 log dynamic range; sensitivity confirmed at the stated minimum detection level; AZT inhibition ≥90% at 50 μM; no-enzyme background below the stated detection limit |
| Key Features | Non-radioactive format replaces traditional [3H]-TTP radiometric RT assays; PCR-PERT format provides sensitivity orders of magnitude greater than standard gel-based detection; applicable for regulatory biopharmaceutical retroviral safety testing; certified positive control allows cross-laboratory activity standardization |
For research use only, not for clinical use.
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