Reverse Transcriptase Assay (PERT) - Biofargo

Description

The Reverse Transcriptase Assay (PERT) is a highly sensitive, one-step real-time qPCR analytical reagent engineered specifically for the quantitative evaluation of reverse transcriptase (RT) activity. Utilizing the Product-Enhanced Reverse Transcriptase (PERT) methodology, this system seamlessly integrates RNA reverse transcription and DNA amplification into a single-tube reaction, enabling the rapid and precise molecular analysis of RT enzymes.

Assay Workflow

1. Sample Preparation:
Target analytes 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 quantitative analysis of RT activity.

2. Signal Analysis:
Continuous fluorescence monitoring allows for the precise evaluation of the amplified target sequences.

Key Features & Benefits

• Wide Sample Applicability:
  Compatible with cell culture supernatant, cell lysate, and purified reverse transcriptase solutions, making it highly suitable for diverse in vitro research and bioprocessing applications.
• Ultra High Sensitivity & Broad Linear Range :
  Capable of evaluating as few as dozens of RT molecules, with a linear analytical range spanning at least six orders of magnitude, ensuring precise target quantification.
• Minimal Interference & Cross-Contamination Prevention :
  One-step sample addition minimizes variability, while the single-tube reaction setup prevents sample cross-interference. DNA polymerases lacking RT activity do not interfere with the molecular evaluation.
• Built-in Positive Control :
  Includes a reverse transcriptase positive control, allowing for straightforward standard curve generation and significantly reducing the likelihood of false-negative signals, ensuring reliable and reproducible data.
• One-Step Protocol :
  Streamlined workflow for rapid and robust RT activity analysis.
• Exceptional Sensitivity :
  Detects extremely low levels of RT with unmatched accuracy.
• Integrated Positive Control :
  Facilitates precise quantification and enhances data reliability.
• High Assay Integrity :
  Ensures consistent and reproducible analytical results in every run.
• 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

Specification and ingredient list

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 quantitative signal analysis, making it fully compatible with standard real-time PCR platforms that support FAM-based fluorescence monitoring, 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