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Updated: May 28, 2026
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Pyrosequencing for Microbial Identification and Characterization
Published on: August 22, 2013
Pyrosequencing on templates generated by asymmetric nucleic acid sequence-based amplification (asymmetric-NASBA)
Huning Jia1, Zhiyao Chen, Haiping Wu
1Department of Analytical Chemistry, China Pharmaceutical University, Nanjing, China. jiahuning@sina.com
The Analyst
|October 21, 2011
Summary
Asymmetric nucleic acid sequence-based amplification (NASBA) significantly boosts single-stranded cDNA for pyrosequencing. This method enhances amplicon sequencing, paving the way for advanced clinical diagnostics.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- Pyrosequencing is a valuable technique for DNA sequence verification.
- Nucleic acid sequence-based amplification (NASBA) is a method for amplifying RNA or DNA.
- Integrating NASBA with pyrosequencing presents challenges in amplicon preparation.
Purpose of the Study:
- To adapt asymmetric NASBA for efficient pyrosequencing of amplicons.
- To optimize NASBA conditions for increased single-stranded cDNA production.
- To evaluate the utility of this combined approach for molecular diagnostics.
Main Methods:
- Development of asymmetric NASBA using unequal primer concentrations.
- Optimization of primer ratios and nucleotide concentrations (dNTPs and NTPs).
- Real-time detection using molecular beacons to quantify single-stranded cDNA.
- Pyrosequencing of amplicons generated by asymmetric NASBA.
Main Results:
- Asymmetric NASBA increased single-stranded cDNA yield by 12-fold compared to conventional NASBA.
- Successful pyrosequencing of over 20 bases from asymmetric NASBA amplicons.
- Human parainfluenza virus (HPIV) was used as a successful amplification template.
- Demonstrated the practicality of combining NASBA with pyrosequencing.
Conclusions:
- Asymmetric NASBA is an effective method to enhance amplicon quality for pyrosequencing.
- The combined NASBA-pyrosequencing system shows significant potential for clinical diagnosis.
- This approach offers a novel strategy for genetic analysis and disease detection.
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