Engineered Janus probes modulate nucleic acid amplification to expand the dynamic range for direct detection of viral genomes in one microliter crude serum samples,Chemical Science

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Engineered Janus probes modulate nucleic acid amplification to expand the dynamic range for direct detection of viral genomes in one microliter crude serum samples
Chemical Science ( IF 7.6 ) Pub Date : 2017-10-27 00:00:00 , DOI: 10.1039/c7sc03994h
Yue Zhao _{1,

2,

3,

4,

5} , Feng Chen _{1,

2,

3,

4,

5} , Jing Qin _{1,

2,

3,

4,

5} , Jing Wei _{1,

2,

3,

4,

5} , Wenhua Wu _{3,

4,

5,

6,

7} , Yongxi Zhao _{1,

2,

3,

4,

5}

Affiliation

The viral genome load in diverse clinical samples varies over several orders of magnitude (e.g. 1–10⁴ copies per μL), thus a dynamic range-extended and sensitive analysis method is highly desired. However, existing well-developed nucleic acid amplification systems always suffer from either a limited dynamic range or modest sensitivity for analysis of these samples. Herein, we propose a general engineered Janus probe to modulate the thermodynamics and kinetic properties of the amplification reaction. Through rational regulation, the Janus system improves the performance by both reducing the background and enhancing the signal, expanding the operative dynamic range by 2 orders of magnitude. This proposed approach achieves a detection limit for hepatitis B virus (HBV) DNA of down to 3 copies and can be successfully applied for direct quantification of the HBV genome in one microliter crude serum samples without any pretreatment. The results are consistent with clinical diagnosis and hold considerable potential to discriminate healthy volunteers and patients at different disease stages. Whereas, following the same operation, the representative well-developed system provided serious false-negative results using such trace amounts of samples from clinically confirmed positive patients.

中文翻译：

工程Janus探针可调节核酸扩增，扩大动态范围，可直接检测一微升粗血清样品中的病毒基因组

不同临床样品中病毒基因组的负载量变化了几个数量级（例如1–10 ⁴每μL拷贝数），因此非常需要一种动态范围扩展且灵敏的分析方法。然而，现有的发达的核酸扩增系统对于这些样品的分析总是受限于有限的动态范围或适度的灵敏度。在本文中，我们提出了一种通用的Janus探针来调节扩增反应的热力学和动力学性质。通过合理的调节，Janus系统可通过减少背景和增强信号，将操作动态范围扩大2个数量级来提高性能。该方法可将乙型肝炎病毒（HBV）DNA的检出限降低至3份，并且无需任何预处理即可成功用于一微升粗血清样品中HBV基因组的直接定量分析。结果与临床诊断是一致的，并且在区分健康志愿者和处于不同疾病阶段的患者方面具有相当大的潜力。而经过相同的操作，具有代表性的发达系统使用来自临床确认阳性患者的痕量样品提供了严重的假阴性结果。

更新日期：2017-11-08

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