Department of Analytical Chemistry, China Pharmaceutical University, Nanjing, China.
Analyst. 2011 Dec 21;136(24):5229-33. doi: 10.1039/c1an15766c. Epub 2011 Oct 19.
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.
焦磷酸测序是验证扩增子序列的理想工具。为了在核酸序列扩增基础上的扩增子（NASBA）上进行焦磷酸测序，提出了 T7 启动子引物和逆转录引物浓度不等的非对称 NASBA。通过优化两种引物的比例以及 dNTP 和 NTP 的浓度，通过实时检测与感兴趣的 cDNA 特异性的分子信标，发现非对称 NASBA 扩增子中单链 cDNA 的量比常规 NASBA 增加了 12 倍。使用副流感病毒（HPIV）作为扩增模板，通过非对称 NASBA 扩增子成功检测到超过 20 个碱基。初步结果表明，NASBA 与焦磷酸测序系统的结合是实用的，应该为临床诊断开辟新的领域。
