Zhang Lutan, Peng Jian, Chen Junman, Xu Lulu, Zhang Yangli, Li Ying, Zhao Jie, Xiang Linguo, Ge Yunsheng, Cheng Wei
The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P. R. China.
United Diagnostic and Research Center for Clinical Genetics, Women and Children's Hospital, School of Medicine & School of Public Health, Xiamen University, Xiamen 361000, P. R. China.
Anal Chem. 2021 Apr 6;93(13):5621-5628. doi: 10.1021/acs.analchem.1c00405. Epub 2021 Mar 25.
Papillary thyroid carcinoma (PTC) is the most common thyroid cancer with high incidence in endocrine tumors, which emphasizes the significance of accurate diagnostics. Still, the commonly used cytological method (fine-needle aspiration (FNA) cytology) and molecular diagnostic methods (such as PCR and sequencing) are limited in terms of diagnostic time, sensitivity, and user-friendliness. In this study, we introduce a novel Zip recombinase polymerase amplification (Z-RPA) strategy to efficiently detect rare mutant alleles in PTC fine-needle aspiration samples, which is sensitive, fast, and simple to manipulate. Using Zip nucleic acid (ZNA) probes to clamp the mutation region, the phi 29 polymerase could selectively displace mismatched ZNA probes and start amplification, while leaving complementary ZNA probes untouched and blocking amplification according to genotype. We demonstrated the good sensitivity and specificity of this strategy with optimized conditions and design, which enabled detection of BRAF V600E mutation in a total 4 ng of genomic DNA within 40 min (≈1 copy). Robust behavior in clinical specimen analysis was also demonstrated. The Z-RPA strategy provides a pragmatic approach to rapidly, sensitively, and easily detect BRAF V600E mutation in clinical fine-needle aspiration samples, which is a promising method for early cancer diagnosis and treatment guideline.
甲状腺乳头状癌（PTC）是内分泌肿瘤中最常见、发病率最高的甲状腺癌，这凸显了准确诊断的重要性。然而，常用的细胞学方法（细针穿刺（FNA）细胞学）和分子诊断方法（如PCR和测序）在诊断时间、灵敏度和用户友好性方面都存在局限性。在本研究中，我们引入了一种新型的Zip重组酶聚合酶扩增（Z-RPA）策略，以有效检测PTC细针穿刺样本中的罕见突变等位基因，该策略灵敏、快速且操作简单。使用Zip核酸（ZNA）探针夹住突变区域，phi 29聚合酶可以选择性地置换错配的ZNA探针并启动扩增，同时保持互补的ZNA探针不变，并根据基因型阻断扩增。我们通过优化条件和设计证明了该策略具有良好的灵敏度和特异性，能够在40分钟内（≈1个拷贝）在总共4 ng的基因组DNA中检测到BRAF V600E突变。在临床标本分析中也展示了其可靠的性能。Z-RPA策略为快速、灵敏且轻松地检测临床细针穿刺样本中的BRAF V600E突变提供了一种实用方法，是早期癌症诊断和治疗指南的一种有前景的方法。
