Clinically and biologically, it is essential to detect rare DNA-sequence variants for early cancer diagnosis or drug-resistance mutation identification. Some of the common quantitative polymerase chain reaction (qPCR)-based variant detection methods are restricted in the limit of detection (LoD) because the DNA polymerases used for these methods have a high polymerase misincorporation rate; thus, the detection sensitivity is sometimes unsatisfactory. With the proofreading activity, high-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. However, there are currently no proper probe-based designs functioning as the fluorescence indicator allowing multiplexed HiFi qPCR reactions, thus restricting the application of HiFi DNA polymerases like the variant detection. We presented the occlusion system, composed of a 5′-overhanged primer with a fluorophore modification and a probe with a short-stem hairpin and a 3′ quencher modification. We demonstrated that the occlusion system allowed multiplexing HiFi qPCR reaction, and it was compatible with the current variant-enrichment method to improve the LoD up to 10-fold. Thus, the occlusion system satisfactorily functioned as an efficient fluorescence indicator in HiFi qPCR reactions and allowed the application of HiFi DNA polymerases in variant detection methods to improve detection sensitivity.

中文翻译：

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发夹结构促进实时聚合酶链式反应中的多重高保真 DNA 扩增

在临床和生物学上，检测罕见的 DNA 序列变异对于早期癌症诊断或耐药突变鉴定至关重要。一些常见的基于定量聚合酶链反应 (qPCR) 的变异检测方法受到检测限 (LoD) 的限制，因为用于这些方法的 DNA 聚合酶具有较高的聚合酶错误掺入率；因此，检测灵敏度有时不能令人满意。通过校对活动，高保真 (HiFi) DN​​A 聚合酶的保真度提高了 50 到 250 倍。然而，目前还没有合适的基于探针的设计作为荧光指示剂，允许多重 HiFi qPCR 反应，从而限制了 HiFi DNA 聚合酶的应用，如变异检测。我们介绍了遮挡系统，由带有荧光团修饰的 5'-悬垂引物和带有短茎发夹和 3' 猝灭剂修饰的探针组成。我们证明了闭塞系统允许多路复用 HiFi qPCR 反应，并且与当前的变体富集方法兼容，可将 LoD 提高 10 倍。因此，闭塞系统在 HiFi qPCR 反应中令人满意地用作有效的荧光指示剂，并允许在变体检测方法中应用 HiFi DNA 聚合酶以提高检测灵敏度。