Abstract
<jats:p>Background/Objectives: Rapid DNA systems accelerate STR profiling but often require the consumption of the entire swab, limiting confirmation testing or downstream analyses. We previously validated a simple subsampling protocol for blood swabs on the RapidHITTM ID, using a rigid subungual mini-swab (Copan Italia S.p.A). A new version of this instrument has recently been released, featuring redesigned software and consumables. The RapidINTELTM Plus sample cartridge now enables two distinct lysis/extraction protocols, expanding analytical possibilities for rich biological traces. We evaluated subsampling performance using the subungual mini-swab and microFLOQ® swabs (Copan Italia S.p.A), and assessed feasibility for both blood and buccal reference swabs. Methods: Whole blood from four donors was deposited onto regular Copan swabs (10 µL) or microFLOQ® swabs (1 µL). A comparison was performed between the direct analysis of blood swabs using a RapidHITTM ID V1 (RapidINTELTM cartridge) and a RapidHITTM ID V2 (RapidINTELTM Plus cartridge, GENERAL protocol). Subsequently, both the GENERAL and SPECIALIZED protocols were tested after subsampling from primary blood or buccal swabs dried for 24 h using either a subungual mini-swab or a microFLOQ®. Results: Blood-swab subsampling on the V2 produced usable STR profiles with both the subungual mini-swab and the microFLOQ®. The subungual mini-swab was compatible with both the GENERAL and SPECIALIZED protocols. For blood applications, microFLOQ® fiber treatment showed no inhibitory effects. Reference buccal swabs were successfully analyzed with the RapidINTELTM Plus cartridge, either directly (regular swab) or via subungual subsampling under both protocols. In contrast, in this feasibility dataset (single analysis per donor per condition), subsampling a reference swab with microFLOQ® did not yield suitable profiles for RapidINTELTM Plus analysis under the tested conditions. Conclusions: This feasibility study indicates that the subsampling strategy can be applied on the RapidHITTM ID V2, particularly using subungual mini-swabs, to retain the primary swab for potential downstream testing while maintaining usable STR profile quality for blood and buccal reference workflows under the tested conditions.</jats:p>