PO.PR01.03 · 预防研究
通过处理器介导PCR实现单反应KRAS变异图谱分析：利用iProbe增强SNV区分能力
Single-reaction KRAS variant profiling via processor-mediated PCR: Enhanced SNV discrimination using the iProbe
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
对单核苷酸变异（SNV）的准确区分仍是定量PCR（qPCR）和数字PCR（dPCR）的一大局限。即使采用优化的水解探针，等位基因间细微的热力学差异往往也会导致交叉反应、在检测限附近敏感性下降，以及需要多个单重反应。这些制约对于需要在紧密相邻的突变位点检测低频变异的肿瘤学应用而言尤为棘手。KRAS密码子12和13的突变是多种实体瘤中临床可操作的驱动因素，影响预后及对靶向治疗的应答。然而，这些变异极度邻近，使得同时进行高特异性检测变得困难；目前大多数基于PCR的检测需要分开的反应，或在相邻等位基因间表现出脱靶信号。我们开发了处理器介导PCR，这是一种检测架构，利用通用"处理器"寡核苷酸和通用荧光探针将靶标扩增与读出解耦。这种化学方法支持高度多重化、降低检测成本，并实现独立于荧光团通道限制的可调节特异性。与此同时，我们设计了一种模块化多结构域探针结构iProbe，其仅在与靶标变异完全杂交时才产生信号，从而实现卓越的SNV区分。为验证该平台，我们创建了一个单管KRAS G12/G13检测，将处理器介导PCR与结构域工程化的iProbe相结合。在dPCR中，该检测同时检测到所有主要的KRAS密码子12和13突变体，且无可检测到的交叉反应。敏感性研究证实，在低至0.01%（1:10,000突变型:野生型）的等位基因频率下仍能可靠检测，并具有清晰的数字分离。搭配iProbe的处理器介导PCR为长期存在的特异性和多重化障碍提供了强有力的解决方案，能够在单个反应中实现对临床相关SNV的经济高效、高选择性检测。
查看英文原文 English abstract
Accurate discrimination of single-nucleotide variants (SNVs) remains a major limitation of quantitative PCR (qPCR) and digital PCR (dPCR). Even with optimized hydrolysis probes, subtle thermodynamic differences between alleles often lead to cross-reactivity, reduced sensitivity near the limit of detection, and the need for multiple singleplex reactions. These constraints are particularly problematic for oncology applications requiring detection of low-frequency variants across closely spaced mutation sites. KRAS mutations in codons 12 and 13 represent clinically actionable drivers in multiple solid tumors, influencing prognosis and response to targeted therapies. However, the extreme proximity of these variants makes simultaneous, highly specific detection difficult; most current PCR-based assays require separate reactions or exhibit off-target signal among neighboring alleles. We developed processor-mediated PCR, a detection architecture that decouples target amplification from readout using a universal “processor” oligo and universal fluorescent probes. This chemistry supports high multiplexing, reduced assay cost, and tunable specificity independent of fluorophore channel limitations. In parallel, we engineered a modular multi-domain probe structure, iProbe, that generates signal only upon complete hybridization to a target variant, enabling exceptional SNV discrimination. To demonstrate this platform, we created a single-tube KRAS G12/G13 assay combining processor-mediated PCR with domain-engineered iProbes. In dPCR, the assay simultaneously detected all major KRAS codon 12 and 13 mutants with no detectable cross-reactivity. Sensitivity studies confirmed reliable detection at allele frequencies down to 0.01% (1:10,000 mutant:wild-type) with clean digital separation. Processor-mediated PCR with iprobes provides a powerful solution to longstanding specificity and multiplexing barriers, enabling cost-effective, highly selective detection of clinically relevant SNVs in a single reaction.
利益披露 Disclosure
E. K. Pomaranski, None..
A. M. McCoy, None..
K. Spork, None..
N. Mertz, None..
V. Makarov, None.