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

Abstract

ABSTRACT 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 > 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.