Reverse Transcriptase Assay (PERT) – Biofargo

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. This assay is designed for rapid, contamination-free detection of RT using a one-step workflow.

This assay employs FAM-based fluorescence detection, making it compatible with qPCR instruments, including the 7500 Fast System. The included PERT Sample Diluent facilitates effective viral sample preparation, enhancing accuracy and reproducibility.

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.

PERT Kit User Manual