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Construction strategy for an internal amplification control for real-time diagnostic assays using nucleic Acid
David Rodríguez-Lázaro1, Martin D'Agostino, Maria Pla
1Institute of Food and Agricultural Technology, University of Girona, Campus Montilivi, s/n 17071 Girona, Spain.
Journal of Clinical Microbiology
|December 8, 2004
Summary
An internal amplification control (IAC) ensures molecular amplification assay validity. This study presents a novel method for constructing RNA-based IACs for real-time nucleic acid sequence-based amplification (NASBA) assays.
Area of Science:
- Molecular Biology
- Biotechnology
- Analytical Chemistry
Background:
- Internal amplification controls (IACs) are crucial for validating molecular amplification assays.
- Current IAC methods may require complex design or lack broad applicability.
Purpose of the Study:
- To develop a generalizable strategy for constructing RNA-based internal amplification controls (IACs).
- To integrate these IACs into molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assays.
Main Methods:
- A two-phase construction process involving DNA template synthesis and in vitro RNA transcription.
- Utilizing T7 RNA polymerase to generate RNA transcripts serving as IACs.
- Coamplification of IACs with target sequences and detection using molecular beacons.
Main Results:
- Successfully constructed and validated RNA-based IACs for NASBA assays.
- Demonstrated the utility of IACs in correctly interpreting diagnostic results from spiked and natural clinical samples.
- Optimized IAC incorporation and performance within the assay.
Conclusions:
- The presented strategy provides a robust method for generating effective RNA-based IACs.
- This approach enhances the reliability and accuracy of molecular amplification-based diagnostic tests.
- The developed IACs are applicable to various NASBA assays, including those for pathogen detection like Mycobacterium avium subsp. paratuberculosis.