Evaluation of an RNA extraction method to detect SARS-CoV-2 RNAemia in postmortem whole blood

RNAemia, defined as the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in blood, is correlated with the severity of coronavirus disease 2019 (COVID-19). Although serum or plasma is commonly used for viral RNA extraction in clinical studies, separating serum in forensic postmortem specimens is often difficult because of postmortem changes. This study aimed to establish an efficient method for extracting SARS-CoV-2 RNA from postmortem whole blood and assess its forensic applicability using actual case specimens. Three extraction methods-acid guanidinium thiocyanate-phenol-chloroform (AGPC), spin column (SC), and magnetic bead method-were compared to identify an efficient approach for SARS-CoV-2 RNA extraction from whole blood. The AGPC method yielded the highest viral RNA recovery, although reproducibility was limited. We therefore evaluated a modified AGPC-SC method, in which whole blood was homogenized, subjected to AGPC phase separation, and purified using the SC method. This procedure maintained the yield of viral RNA while improving reproducibility. The optimized method showed a positive percent agreement of 85.3% and a negative percent agreement of 92.5% for postmortem whole blood samples from COVID-19 cases compared with serum. Whole blood and serum viral RNA loads were strongly correlated (Spearman's ρ = 0.9014), with a small mean difference on Bland-Altman analysis (-0.053 log₁₀ copies/µL). Thus, postmortem whole blood may serve as an alternative to serum, and the AGPC-SC method involving homogenization provides a practical approach for SARS-CoV-2 RNA extraction in forensic practice.