Description

The One-Step Product-Enhanced Reverse Transcriptase (PERT) Assay is a highly sensitive and quantitative method for detecting reverse transcriptase (RT) activity in biological samples, particularly in retroviral research, gene therapy, and exosome studies.

Complying with World Health Organization (WHO) and U.S. Centers for Disease Control and Prevention (CDC) guidance, which stipulate that biological products must be free of retrovirus contamination, this assay provides a robust solution for confirming the absence of retroviral RT activity. The method follows pharmacopeial PERT principles and enables direct, one-tube real-time detection of RT, ensuring rapid and contamination-free results.

This assay employs FAM-based fluorescence detection, making it fully compatible with standard qPCR instruments such as the 7500 Fast System. The included PERT Sample Diluent ensures efficient viral sample preparation, improving accuracy and reproducibility for high-throughput applications in research and quality control.

Assay Workflow

1. Sample Preparation:
Viral samples or exosomes are resuspended in PERT Sample Diluent.

2. Reverse Transcription & Amplification:
The one-step assay simultaneously reverse transcribes RNA and amplifies DNA, enabling real-time RT activity detection.

3. Detection:
Fluorescence signal from FAM-labeled probes provides quantitative RT activity measurement.

Key Features & Benefits

Wide Sample Applicability :
Compatible with cell supernatant, cell lysate, and purified reverse transcriptase solution, making it suitable for diverse research applications.
Ultra High Sensitivity & Broad Linear Range :
Capable of detecting as few as dozens of RT molecules, with a linear detection range spanning at least six orders of magnitude, ensuring precise quantification.
Minimal Interference & Cross-Contamination Prevention :
One-step sample addition minimizes variability, while the single-tube reaction setup eliminates cross-contamination risks. DNA polymerase without RT activity does not interfere with detection.
Built-in Positive Control :
Includes a reverse transcriptase positive control, allowing for easy standard curve generation and significantly reducing the likelihood of false-negative results, ensuring reliable and reproducible data.
One-Step Protocol :
Streamlined workflow for faster, contamination-free RT detection.
Exceptional Sensitivity :
Detects extremely low levels of RT with unmatched accuracy.
Integrated Positive Control :
Facilitates precise quantification and enhances data reliability.
No Cross-Contamination :
Ensures clean and reproducible results in every assay.

Applications

Retrovirus and Lentivirus Detection :
Validate virus production and monitor RT enzyme activity.
Gene Therapy & Vaccine Development :
Ensure the safety and efficiency of viral vectors.
Exosome Research :
Measure RT activity in exosomal cargo for diagnostic and biomarker studies.
Quality Control for RT Enzymes :
Assess RT purity and performance in molecular biology applications.
Medical Diagnostics :
Detection of reverse transcriptase contamination in drug formulations.
Industrial Production :
Quality inspection of reverse transcriptase enzyme manufacturing.
Cell Culture Research :
Detection of RT activity in cultured cells to monitor potential contamination.
Food Safety & Inspection :
Screening for reverse transcriptase contamination in food products.

Kit Specifications

Detection Method	FAM-based qPCR
Format	One-step RT-PCR
Reaction Volume	20µL
Kit Contents	Sufficient for 50 reactions
Reverse Transcriptase Sensitivity	Capable of detecting low levels of RT (~200,000 U/mg MMLV RT activity)

Specification and ingredient list

Components	No.	Specification	Packaging
PERT sample dilution	221044a	12 mL	30 mL natural-colored bottle
MMLV reverse transcriptase (200 U/μL)	11-220628	20 μL	0.5 mL red-capped tube
PERT primer-probe mixture	15-221044ba	50 times	0.5 mL brown plastic tube
PERT RNA template solution	221044v2	50 times	0.5 mL white-capped tube
2×PERT MasterMix	221044b	500 μL	0.5 mL green-capped tube
User manual	221044sc	1 copy	None

Reverse Transcriptase Detection Assay - FAQ

1. Is this assay kit SYBR Green- or TaqMan-based? Can we use it with the 7500 Fast System?

The assay utilizes a FAM channel for detection, making it compatible with qPCR systems that support FAM-based fluorescence detection, including the 7500 Fast System.

2. Is this a one-step or two-step PERT assay?

This is a one-step PERT assay, which simplifies the workflow and reduces the risk of contamination.

3. Does the assay include a lysis buffer for viral sample preparation?

The kit includes a PERT Sample Diluent, which is designed for resuspending virus pellets and preparing cell lysates. If additional lysis buffer is required for specific sample types, we can recommend suitable options upon request.

4. What is the sensitivity of the assay?

The assay is highly sensitive, capable of detecting only a few dozen molecules of reverse transcriptase. The included MMLV Reverse Transcriptase has a specific activity of approximately 200,000 U/mg.

5. How many reactions does the kit provide?

The kit contains enough reagents for 50 reactions, with a standard reaction volume of 20µL per assay.

References:

Draft guidance for industry: Characterization and Qualification of Cell Substrates and Other Biological Starting Materials Used In The Production of Viral Vaccines for the Prevention and Treatment Of Infectious Diseases: SSeptember, 2006
Draft Gguideline On Virus Safety Evaluation Of Biotechnological:Investigational Medicinal Products. European Medicines Agency: Evaluation of Mmedicines for human use, Doc. Ref. EMEA/CHMP/BWP/398498/2005-CORR, 28 June 2006
ICH Harmonized Tripartite Guideline: Viral Safety Evaluation Of Biotechnology Products Derived From Cell Lines Of Human Or Animal Origin; Q5a(r1): DATED 23 SSeptember 1999
Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use; February 28,1997
Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)
CBER Letter to Industry 1998

PERT Kit User Manual

Reverse Transcriptase Assay (PERT) - Biofargo