Phosphorothioated Primers Lead to Loop-Mediated Isothermal Amplification at Low Temperatures
Phosphorothioated Primers Lead to Loop-Mediated Isothermal Amplification at Low Temperatures
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DOI：
10.1021/acs.analchem.8b02062
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发表时间：
2018-07-17
影响因子：
7.4
通讯作者：
Ellington, Andrew D.
中科院分区：
文献类型：
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作者：
Cai, Sheng;Jung, Cheulhee;Ellington, Andrew D.
环介导等温扩增（LAMP）是一种非常强大的核酸检测工具，具有高灵敏度和特异性。然而，LAMP在65摄氏度左右显示出最佳性能，这限制了在护理点测试（POCT）中的应用。在这里，我们已经开发了一种使用硫代磷酸化引物（PS-LAMP）的LAMP版本，以在生长的多联体末端实现更有效的发夹形成和延伸，因此在低得多的温度下工作。通过包括诸如离液剂（尿素）和单链DNA结合蛋白（SSB）的额外因素，在40 ° C下用PS-LAMP进行扩增子检测的灵敏度和选择性与在65 ° C下的常规LAMP反应相当。
Loop-mediated isothermal amplification (LAMP) is an extremely powerful tool for the detection of nucleic acids with high sensitivity and specificity. However, LAMP shows optimal performance at around 65 degrees C, which limits applications in point-of-care-testing (POCT). Here, we have developed a version of LAMP that uses phosphorothioated primers (PS-LAMP) to enable more efficient hairpin formation and extension at the termini of growing concatamers, and that therefore works at much lower temperatures. By including additional factors such as chaotropes (urea) and single-stranded DNA binding protein (SSB), the sensitivities and selectivities for amplicon detection with PS-LAMP at 40 degrees C were comparable with a regular LAMP reaction at 65 degrees C.