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Non-amplification nucleic acid detection with thio-NAD cycling
Sou Yamura1, Naoki Kawada1, Shinnosuke Yamakado1
1Department of Biology, Waseda University, Shinjuku, Tokyo, Japan.
Journal of Microbiological Methods
|December 10, 2022
Summary
This study introduces a novel non-amplification nucleic acid detection method combining probe hybridization and thio-NAD cycling. This technique accurately detects the Mycobacterium tuberculosis mpt64 gene while avoiding amplification errors.
Area of Science:
- Molecular Biology
- Biotechnology
- Diagnostic Assays
Background:
- Polymerase Chain Reaction (PCR) is vital in biology and medicine but prone to false positives/negatives.
- A non-amplification nucleic acid detection method is needed to overcome PCR limitations.
Purpose of the Study:
- To develop and validate a novel nucleic acid detection method without amplification.
- To apply this method for detecting the Mycobacterium tuberculosis mpt64 gene.
Main Methods:
- Nucleic acid probe hybridization coupled with thio-NAD cycling.
- Utilized two cDNA probes targeting the mpt64 gene, one for immobilization and another linked to alkaline phosphatase (ALP).
- ALP hydrolyzed a substrate, initiating a dehydrogenase-driven cycling reaction with thio-NAD and NADH.
Main Results:
- Successfully detected single-stranded DNA, double-stranded DNA, plasmid DNA, and whole M. tuberculosis BCG genomes.
- Detection limit achieved at 10^5-10^6 copies/assay.
- Non-tuberculosis mycobacteria species were below the detection limit, demonstrating specificity.
Conclusions:
- The developed method offers a reliable alternative to amplification-based nucleic acid detection.
- This approach mitigates errors associated with PCR, enhancing diagnostic accuracy.
- The method shows high specificity for Mycobacterium tuberculosis detection.
