重组酶聚合酶扩增
分子生物学
甲状腺结节
化学
冷PCR
核酸
聚合酶链反应
聚合酶
DNA
甲状腺癌
肽核酸
癌症研究
癌症
突变
点突变
遗传学
甲状腺
生物
基因
生物化学
作者
Lutan Zhang,Jian Peng,Junman Chen,Lulu Xu,Yanli Zhang,Ying Li,Zhao Jie,Linguo Xiang,Yunsheng Ge,Wei Cheng
标识
DOI:10.1021/acs.analchem.1c00405
摘要
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.
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