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One-step fluorescent probe product-enhanced reverse transcriptase assay
B A Arnold1, R W Hepler, P M Keller
1Department of Virus and Cell Biology, Merck Research Laboratories, West Point, PA 19486-0004, USA.
Biotechniques
|July 21, 1998
Summary
A new fluorescent probe assay enhances reverse transcriptase (RT) detection. This one-step method is faster and highly sensitive for identifying retroviral infections and characterizing RT activity.
Area of Science:
- Biotechnology
- Molecular Biology
- Virology
Background:
- Conventional reverse transcriptase (RT) assays lack sensitivity for retroviral detection.
- Existing PCR-based RT assays are sensitive but labor- and time-intensive.
- The product-enhanced reverse transcriptase (PERT) assay requires a two-step process with Southern blot or ELISA detection.
Purpose of the Study:
- To develop a faster, more efficient RT assay.
- To improve the high-throughput detection of retroviral infections.
- To enhance the characterization of RT activity.
Main Methods:
- Modification of the PERT assay into a one-step, fluorescent probe-based method.
- Utilizing real-time PCR with fluorescent probes for direct detection and quantitation.
- Assay completion from sample dilution to results in approximately 5 hours.
Main Results:
- Achieved a one-step, fluorescent probe-based RT assay.
- Maintained assay sensitivity and specificity compared to previous methods.
- Demonstrated a dynamic range of 6 logs, reducing the need for extensive sample dilution.
- Assay completed in approximately 5 hours.
Conclusions:
- The enhanced fluorescent PERT assay offers a rapid and sensitive method for RT detection.
- This assay is suitable for high-throughput screening of retroviral infections.
- The assay facilitates efficient characterization of RT activity.