Enzymatic and chemical-based methods to inactivate endogenous blood ribonucleases for nucleic acid diagnostics.

Abstract There are ongoing research efforts into simple and low-cost point-of-care (POC) 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. Here, we investigate alternatives to standard guanidinium-based methods for RNase inactivation using an activity assay with an RNA substrate that fluoresces when cleaved. We report on the effects of proteinase K, nonionic surfactants, sodium dodecyl sulfate (SDS), dithiothreitol (DTT), and other additives on RNase activity in human serum. While proteinase K has been widely used in protocols for nuclease inactivation, we found that high concentrations of proteinase K are unable to eliminate RNase activity in serum, unless used in concert with denaturing concentrations of SDS. We observed that SDS must be combined with proteinase K, DTT, or both for irreversible and complete RNase inactivation in serum. Our work provides an alternative chemistry for inactivating endogenous RNases for use in simple, low-cost POC NATs for bloodborne pathogens.