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One-step colorimetric genotyping of single nucleotide polymorphism using probe-enhanced loop-mediated isothermal amplification (PE-LAMP).
Theranostics ( IF 12.4 ) Pub Date : 2019-01-01 , DOI: 10.7150/thno.33980
Sheng Ding 1, 2 , Rong Chen 3 , Gangyi Chen 1 , Mei Li 1 , Jiayu Wang 1, 2 , Jiawei Zou 1 , Feng Du 1 , Juan Dong 1 , Xin Cui 1 , Xin Huang 1 , Yun Deng 3 , Zhuo Tang 1
Affiliation  

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.

中文翻译:

使用探针增强的环介导的等温扩增(PE-LAMP)进行单核苷酸多态性的一步式比色基因分型。

单核苷酸多态性(SNP)是与许多生理和病理表型相关的最丰富的分子标记。恒温,准确且具有成本效益的SNP检测可以在现场即时检测(POCT)或现场诊断中产生巨大的差异。然而,现场SNP检测的发展面临着两个挑战,昂贵的仪器和劳动密集的过程。我们报道了一种基于探针增强环介导的等温扩增(PE-LAMP)的新型SNP分型方法,该方法将寡核苷酸探针与常规LAMP结合起来,通过分析扩增效率的巨大差异来实现SNP识别。方法:我们首先将特异性探针的杂交与LAMP的强大扩增相结合,构建了基因分型方法。然后,我们通过对SNP rs3741219进行基因分型来验证该方法,并力求实现一步式可视化打字。最后,我们通过对CYP2C19 * 2和MDR1 C3435T进行基因分型,将该方法应用于药物基因组学测试。结果:成功构建了PE-LAMP来检测SNP,我们的方法灵敏度低至1000拷贝目标DNA,足以进行常规诊断。可以实现在存在过量野生型等位基因时检测突变体的高特异性。通过键入简单处理的唾液样本,它在帮助预测抗血小板药物氯吡格雷的个体反应方面显示出良好的性能。结论:所提出的方法是一步法,比色法,特异性和灵敏性,足以检测未处理的样品,在药物基因组学研究和POCT使用中显示出巨大的潜力。
更新日期:2019-01-01
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