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.

目前正在进行简单且低成本的即时核酸扩增测试 (NAT) 的研究工作，以解决资源有限的临床环境中广泛的诊断需求。血液样本中 RNA 靶标的核酸检测通常需要样品制备，以灭活可快速降解外源性 RNA 的强大的血液核糖核酸酶 (RNase)。大多数 NAT 依赖于使用高浓度胍盐溶解病原体和灭活 RNases 的数十年历史。在此，我们使用活性测定法研究了基于标准胍的 RNase 灭活方法的替代方法，该方法使用切割时会发出荧光的 RNA 底物。报告了蛋白酶 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 已广泛用于核酸酶灭活实验方案，但发现高浓度的蛋白酶 K 无法消除血清中的 RNase 活性，除非与变性浓度的 SDS 一起使用。据观察，SDS 必须与蛋白酶 K、二硫苏糖醇或两者结合使用，才能使血清中的 RNase 不可逆和完全失活。这项工作提供了一种用于灭活内源性 RNase 的替代化学方法，用于血源性病原体的简单、低成本的护理点 NAT。或两者都用于血清中不可逆和完全 RNase 灭活。这项工作提供了一种用于灭活内源性 RNases 的替代化学方法，用于血源性病原体的简单、低成本的护理点 NAT。或两者都用于血清中不可逆和完全 RNase 灭活。这项工作提供了一种用于灭活内源性 RNase 的替代化学方法，用于血源性病原体的简单、低成本的护理点 NAT。