One-step colorimetric genotyping of single nucleotide polymorphism using probe-enhanced loop-mediated isothermal amplification (PE-LAMP)
One-step colorimetric genotyping of single nucleotide polymorphism using probe-enhanced loop-mediated isothermal amplification (PE-LAMP)
复制标题
使用探针增强环介导等温扩增 (PE-LAMP) 对单核苷酸多态性进行一步比色基因分型
DOI：
10.7150/thno.33980
复制
发表时间：
2019-01-01
期刊：
影响因子：
12.4
通讯作者：
Tang, Zhuo
中科院分区：
文献类型：
--
作者：
Ding, Sheng;Chen, Rong;Tang, Zhuo
单核苷酸多态性（single nucleotide polymorphism，SNP）是与多种生理和病理表型相关的最丰富的分子标记。等温、准确且具有成本效益的SNP检测可以在即时检测（POCT）或现场诊断中产生很大的差异。然而，SNP现场检测技术的发展面临着昂贵的仪器和劳动密集型的过程两大挑战。我们报道了一种基于探针增强环介导等温扩增（probe-enhanced loop-mediated isothermal amplification，PE-LAMP）的SNP分型新方法，该方法将寡核苷酸探针与常规LAMP结合，通过分析扩增效率的巨大差异来实现SNP分型。方法：首次建立了特异性探针杂交与LAMP强扩增相结合的基因分型方法。然后通过SNP rs3741219基因分型对该方法进行了验证，并试图实现一步可视化分型。最后，我们将该方法应用于药物基因组学检测，对CYP 2C 19 *2和MDR 1 C3435 T进行基因分型。结果如下：成功构建了检测SNP的PE-LAMP方法，其灵敏度可达1000拷贝，足以满足常规诊断的需要。在过量野生型等位基因存在的情况下，可以实现检测突变体的高特异性。它已显示出良好的性能，在帮助预测抗血小板药物氯吡格雷的个人反应，通过简单处理的唾液样本分型。结论：该方法具有一步法、比色法、特异性强、灵敏度高等优点，可用于药物基因组学研究和POCT应用。
Single nucleotide polymorphism (SNP) is the most abundant molecular marker associated with many physiologic and pathologic phenotypes. An isothermal, accurate and cost-effective SNP detection could make a great difference in point-of-care testing (POCT) or on-site diagnosis. However, there are two challenges, the expensive instrument and labor-intensive process, faced by the development of on-site SNP detection. We reported a novel SNP typing method based on the probe-enhanced loop-mediated isothermal amplification (PE-LAMP), which combines the oligonucleotide probe with a conventional LAMP to realize the SNP discrimination by analyzing the great discrepancy in amplification efficiency. Methods: We firstly constructed the genotyping method by combining the hybridization of the specific probe with the powerful amplification of LAMP. Then we validated the method by genotyping the SNP rs3741219 and we sought to realize one-step visualized typing. Finally, we applied the method to pharmacogenomic testing by genotyping CYP2C19*2 and MDR1 C3435T. Results: The PE-LAMP was successfully constructed to detect SNP and the sensitivity of our method is as low as 1000 copies of target DNA, which is sufficient to routine diagnosis. The high specificity in detecting mutant in the presence of excess wild-type allele could be achieved. It has shown good performance in helping predict the individual response of antiplatelet drug Clopidogrel through typing simply treated saliva samples. Conclusions: The proposed method is one-step, colorimetric, specific and sensitive enough to detect crudely treated samples, showing great potential in the pharmacogenomic study and POCT use.