Development of a Direct Cell-to-PCR Lysis Buffer Using Optimized Non-Ionic Detergents for RNA-Extraction-Free RT-qPCR.

Column-based RNA extraction is time-consuming, involves cumulative losses during transfer, wash, and elution steps, and has reduced recovery from sub-microliter inputs. These limitations restrict gene-expression analysis in small cell populations and low-titre pathogen detection in clinical specimens. We developed the Direct Cell-to-PCR Lysis Buffer, a defined non-ionic detergent formulation containing Tween 20 (0.3%), Triton X-100 (0.1%), and NP-40 (0.1%), for RNA-extraction-free one-step RT-qPCR. Lysates are added directly to reactions without pretreatment, heating, or column purification. Non-ionic detergents produced Ct values comparable to detergent-free controls, whereas SDS, Sarkosyl, and sodium deoxycholate abolished amplification. The method was benchmarked against the standard column-based PureLink RNA Mini Kit using SaOS-2 cells, MCF7 cells, peripheral blood mononuclear cells, and nasal and throat swabs. Direct lysates showed Ct offsets of 0.6 to 2.85 cycles compared with purified RNA, depending on sample type. Ubiquitin-C mRNA was detected down to a single-cell-equivalent input, and plasmid templates were detected to approximately 100 copies per reaction. The buffer was compatible with multiplex ScriptTaq COVID PCR, with unchanged RdRP, N, and RPP30 Ct values, and tolerated sodium azide up to 0.1%, supporting rapid direct RT-qPCR workflows.