Department of Mechanical Engineering, University of Washington, Seattle, Washington.
PATH, Seattle, Washington.
J Mol Diagn. 2020 Aug;22(8):1030-1040. doi: 10.1016/j.jmoldx.2020.04.211. Epub 2020 May 22.
There are ongoing research efforts into simple and low-cost point-of-care nucleic acid amplification tests (NATs) addressing widespread diagnostic needs in resource-limited clinical settings. Nucleic acid testing for RNA targets in blood specimens typically requires sample preparation that inactivates robust blood ribonucleases (RNases) that can rapidly degrade exogenous RNA. Most NATs rely on decades-old methods that lyse pathogens and inactivate RNases with high concentrations of guanidinium salts. Herein, we investigate alternatives to standard guanidinium-based methods for RNase inactivation using an activity assay with an RNA substrate that fluoresces when cleaved. The effects of proteinase K, nonionic surfactants, SDS, dithiothreitol, and other additives on RNase activity in human serum are reported. Although proteinase K has been widely used in protocols for nuclease inactivation, it was found that high concentrations of proteinase K are unable to eliminate RNase activity in serum, unless used in concert with denaturing concentrations of SDS. It was observed that SDS must be combined with proteinase K, dithiothreitol, or both for irreversible and complete RNase inactivation in serum. This work provides an alternative chemistry for inactivating endogenous RNases for use in simple, low-cost point-of-care NATs for blood-borne pathogens.
目前正在进行研究工作，以开发简单且低成本的即时核酸扩增检测（NAT）技术，以满足资源有限的临床环境中的广泛诊断需求。用于血液样本中 RNA 靶标的核酸检测通常需要样品制备，该制备过程可以灭活强大的血液核糖核酸酶（RNase），这些酶可以迅速降解外源性 RNA。大多数 NAT 依赖于数十年前的方法，这些方法使用高浓度的胍盐裂解病原体并使 RNase 失活。在此，我们研究了使用带有可切割荧光 RNA 底物的活性测定法来替代基于标准胍的 RNase 失活方法。报道了蛋白酶 K、非离子表面活性剂、SDS、二硫苏糖醇和其他添加剂对人血清中 RNase 活性的影响。尽管蛋白酶 K 已广泛用于核酸酶失活的方案中，但发现高浓度的蛋白酶 K 无法消除血清中的 RNase 活性，除非与 SDS 的变性浓度一起使用。观察到 SDS 必须与蛋白酶 K、二硫苏糖醇或两者结合使用，才能在血清中不可逆且完全失活 RNase。这项工作为用于血液传播病原体的简单、低成本即时护理 NAT 提供了一种替代的内源性 RNase 失活化学方法。
