期刊
ANALYST
卷 136, 期 24, 页码 5229-5233出版社
ROYAL SOC CHEMISTRY
DOI: 10.1039/c1an15766c
关键词
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资金
- National Natural Science Foundation of China [20975113]
- Science & Technology Development Project of Jiangsu Province [BE2010604]
- Young Teacher Foundation of China Pharmaceutical University
- Central Research Laboratory of Hitachi Ltd, Japan
Pyrosequencing is an ideal tool for verifying the sequence of amplicons. To enable pyrosequencing on amplicons from nucleic acid sequence-based amplification (NASBA), asymmetric NASBA with unequal concentrations of T7 promoter primer and reverse transcription primer was proposed. By optimizing the ratio of two primers and the concentration of dNTPs and NTPs, the amount of single-stranded cDNA in the amplicons from asymmetric NASBA was found increased 12 times more than the conventional NASBA through the real-time detection of a molecular beacon specific to cDNA of interest. More than 20 bases have been successfully detected by pyrosequencing on amplicons from asymmetric NASBA using Human parainfluenza virus (HPIV) as an amplification template. The primary results indicate that the combination of NASBA with a pyrosequencing system is practical, and should open a new field in clinical diagnosis.
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