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$763.00
Sku: 221044
Availability:
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In stock & estimated to ship in 1-2 days by October 30, 2026
Description
Cell banks used to manufacture biologics, vaccines and cell or gene therapy products must be tested for adventitious retroviruses. Because every retrovirus carries reverse transcriptase (RT), measuring RT activity is a broad, sequence-independent way to screen for both known and unknown retroviral particles.
The Biofargo Reverse Transcriptase Assay (PERT, Cat. No. 221044) is a one-step, real-time qPCR kit based on the Product-Enhanced Reverse Transcriptase (PERT) method. RT present in the sample converts a supplied RNA template into cDNA, which is then amplified and detected with a FAM-labeled probe in the same tube. The assay can detect as few as several dozen RT molecules and has a linear range of at least six orders of magnitude.
The kit includes an MMLV reverse transcriptase positive control for building a standard curve in every run, so results can be reported as RT activity rather than a simple positive/negative call.
Specifications
| Cat. No. | 221044 |
| Size | 50 reactions |
| Method | One-step PERT (RT + qPCR in a single tube) |
| Detection Channel | FAM (probe-based) |
| Reaction Volume | 20 µL |
| Sample Types | Ultracentrifuged cell culture supernatant, cell lysate, purified RT solutions |
| Sample Input | 1–5 µL concentrated supernatant, or 0.1–1 µg total protein from cell lysate |
| Sensitivity | Several dozen RT molecules |
| Linear Range | ≥6 orders of magnitude |
| Positive Control | MMLV RT, 200 U/µL (specific activity ~200,000 U/mg) |
| Compatible Instruments | Real-time PCR systems with a FAM channel (e.g., 7500 Fast) |
| Storage | −20°C, 12 months |
| Shipping | Dry ice |
| Grade | Research Use Only |
Kit Components
| Component | Part No. | Quantity | Packaging |
|---|---|---|---|
| PERT Sample Diluent | 221044a | 12 mL | 30 mL natural bottle |
| MMLV Reverse Transcriptase (200 U/µL) | 11-220628 | 20 µL | 0.5 mL red-cap tube |
| PERT Primer-Probe Mix | 15-221044ba | 50 reactions | 0.5 mL brown tube |
| PERT RNA Template Solution | 221044v2 | 50 reactions | 0.5 mL white-cap tube |
| 2× PERT MasterMix | 221044b | 500 µL | 0.5 mL green-cap tube |
| User Manual | 221044sc | 1 copy | — |
Features
- Broad retrovirus screening — detects RT activity regardless of viral sequence, including unknown or endogenous retroviruses.
- High sensitivity — several dozen RT molecules, with at least six logs of linear range.
- Quantitative results — built-in MMLV RT standard curve converts Ct values into RT activity.
- One-step, closed-tube format — RT and amplification in one tube reduces handling and carryover risk.
- No interference from DNA polymerase — DNA polymerases without RT activity do not generate signal.
- Flexible sample preparation — supports ultracentrifuged supernatant or cell lysate.
Application: MCB and WCB Retrovirus Testing
Master Cell Banks (MCB) and Working Cell Banks (WCB) of CHO, HEK293, Vero, hybridoma and other production cell lines are routinely screened for retroviral contamination as part of cell bank characterization (ICH Q5A). The PERT assay is widely used for this purpose because it detects the RT enzyme common to all retroviruses.
Option 1 — Concentrated Supernatant (recommended)
- Collect cell culture supernatant.
- Centrifuge at 11,000 × g for 10 minutes to remove cells and debris.
- Filter through a 0.45 µm membrane.
- Ultracentrifuge at 100,000 × g for 60 minutes to pellet viral particles.
- Resuspend the pellet in PERT Sample Diluent and keep on ice.
- Use 1–5 µL per reaction.
Option 2 — Cell Lysate (no ultracentrifuge needed)
- Pellet 1 × 10⁶ cells.
- Resuspend in 200 µL PERT Sample Diluent.
- Incubate on ice for 20 minutes to lyse.
- Measure total protein with any total protein assay.
- Use 0.1–1 µg total protein per reaction.
Without an ultracentrifuge: do not test unconcentrated supernatant directly. Use the cell lysate method, or concentrate supernatant with 100 kDa MWCO centrifugal ultrafiltration and test the retentate. Skipping concentration can lower sensitivity (roughly 2–3 Ct cycles) and increase the risk of false negatives. Ultrafiltration has not been validated by Biofargo; confirm recovery for your own process.
Run Controls
- Standard curve: 10-fold serial dilutions of the MMLV RT control (dilutions 3–8).
- NTC (no template control): water instead of template.
- NSC (no sample control): PERT Sample Diluent instead of sample, to rule out background RT activity.
- A run is invalid if NTC or NSC shows a typical amplification curve with Ct < 35, or if the standards do not amplify.
For Regulated Programs
This kit is Research Use Only. Data supporting IND/BLA submissions require your own method validation, including recovery of the concentration step, matrix interference testing with your culture medium, and NSC controls in every run. A side-by-side comparison of ultracentrifuged and alternative preparations is recommended when changing methods.
Other Applications
- Viral vector and vaccine manufacturing — retrovirus safety testing of cell substrates and harvests.
- Retrovirus and lentivirus research — monitor virus production and RT activity.
- Exosome research — measure RT activity in extracellular vesicle preparations.
- RT enzyme QC — quantify activity and purity of reverse transcriptase preparations.
- Cell line monitoring — screen research cell lines for retroviral contamination.
Thermal Cycling Program
| Step | Temperature | Time | Cycles |
|---|---|---|---|
| Reverse transcription | 42°C | 10 min | 1 |
| Initial denaturation | 95°C | 5 min | 1 |
| Denaturation | 95°C | 15 s | 35 |
| Annealing (FAM read) | 52°C | 30 s | |
| Extension | 72°C | 30 s |
FAQ
Is this assay probe-based or SYBR Green? Does it work on a 7500 Fast?
It is probe-based with FAM detection and runs on any real-time PCR system with a FAM channel, including the 7500 Fast.
Is it one-step or two-step?
One-step. Reverse transcription and amplification happen in the same tube.
Can I test cell culture supernatant without ultracentrifugation?
Testing unconcentrated supernatant is not recommended because retroviral levels may fall below the detection limit. Use the cell lysate method (1 × 10⁶ cells in 200 µL PERT Sample Diluent) or concentrate with 100 kDa MWCO ultrafiltration.
How much sensitivity is lost without ultracentrifugation?
Expect roughly 2–3 Ct cycles lower sensitivity, which increases the risk of false negatives for low-titer samples.
Does the kit include a lysis buffer?
PERT Sample Diluent is used both to resuspend virus pellets and to lyse cells (20 minutes on ice).
Can this kit be used for MCB/WCB qualification?
Yes, for research and internal testing. It is Research Use Only and not GMP/GLP-grade, so regulatory submissions require your own analytical method validation.
How many reactions are in the kit?
50 reactions of 20 µL.
How should the kit be stored and shipped?
Store at −20°C (12-month shelf life). The kit ships on dry ice.
Related Reading
PERT Testing for MCB/WCB Without an Ultracentrifuge: Sample Preparation Options
References
- ICH Q5A(R1): Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin (1999).
- FDA Draft Guidance: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications (2006).
- EMEA/CHMP/BWP/398498/2005: Guideline on Virus Safety Evaluation of Biotechnological Investigational Medicinal Products (2006).
- FDA: Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1997).
- FDA: Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993).
Download the PERT Kit User Manual (PDF)
For Research Use Only. Not for use in diagnostic procedures.
When can I expect my order to ship?
Most orders are filled and shipped within 2-3 business days from the time they are received.
Our standard shipping usually take 2-5 days.
We also provide express shippping for time-sensitive deliveries.
Email contact@biofargo.com if you have any requirements.
