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Identifying the type(s) of body fluid present in forensic casework exhibits can assist with scene reconstruction and indicate potential activity types. Confirmatory body fluid identification can be achieved using mRNA-based profiling assays. This commonly involves endpoint reverse-transcription PCR (RT-PCR) with capillary electrophoresis (CE) detection. In comparison, real-time quantitative RT-qPCR is more sensitive, quantitative, and does not require post-PCR processing. We have developed real-time RT-qPCR assays for forensic body fluid identification. This study compared the performance (sensitivity, PCR efficiency, precision) of five real-time RT-qPCR kits across circulatory blood, buccal, semen, and vaginal fluid samples with normal and extended (3-step) PCR cycling. An objective scoring system for the experimental performance parameters was considered along with other features of the commercial kits. Statistical analysis by ANOVA and post-hoc Tukey of the slope estimates, which relate to PCR efficiency, revealed that many observed differences were insignificant ( p  &gt; 0.05). Sensitivity and precision were also similar across most kits and PCR cycling protocols. Using the scoring system, the five highest performing kit and cycling combinations were: TaqMan-extended, followed by the TaqPath-normal, QuantiNova-extended, QuantiNova-extended, then UltraPlex-normal. Based upon high performance, room temperature set-up, and multiplexing capability, the UltraPlex kit was selected for our body fluid identification assays.

A comparative study of commercial real-time reverse transcription PCR kits for forensic body fluid identification