Research Article| Volume 64, 102856, May 2023

One-step endpoint RT-PCR assays for confirmatory body fluid identification

  • Author Footnotes
    Courtney Lynch
    Forensic Science Programme, School of Chemical Sciences, The University of Auckland, Auckland, New Zealand

    Forensic Research and Development Team, Institute of Environmental Science and Research Ltd, Auckland, New Zealand
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  • Author Footnotes
    Rachel Fleming
    Correspondence to: Institute of Environmental Science and Research Ltd.,120 Mt Albert Road, Auckland 1025, New Zealand.
    Forensic Research and Development Team, Institute of Environmental Science and Research Ltd, Auckland, New Zealand
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  • Author Footnotes


      • One-step RT-PCR assays were developed for confirmatory body fluid identification.
      • Sensitivity and specificity across body fluids was assessed.
      • ROC curves were used to determine specific peak height cut-offs.
      • The assays successfully detected body fluid mixture components.


      Messenger RNA (mRNA) expression analysis is increasingly used in casework, in the form of multiplex two-step reverse transcriptase PCR (RT-PCR) assays such as CellTyper 2 (CT2), developed by the Institute of Environmental Science and Research (ESR). This paper presents the development of a one-step endpoint RT-PCR workflow to improve the efficiency and precision of confirmatory body fluid identification. A comparative study of commercial one-step RT-PCR kits was undertaken, with the highest performing kit (RNA to CT) retained for further development.
      Sensitivity, specificity across body fluids, and precision was assessed simultaneously using receiver operating characteristic (ROC) curves. An optimal RFU cut-off value which maximised sensitivity and specificity was determined for each marker. All assays performed significantly better when compared to the equivalent of a completely uninformative test (area under the curve of 0.5) for their target body fluid. Sensitivity varied between different donors, but the limit of detectionss were estimated as follows; saliva markers HTN3: 1 in 100 dilution of a whole buccal swab and FDCSP: 1 in 10 dilution of a whole buccal swab, circulatory blood marker SLC4A1: 0.1 µL blood, menstrual fluid markers STC1, MMP10: 1 in 10 dilution of a whole menstrual swab, spermatozoa markers PRM1, TNP1: 0.1 µL semen, seminal fluid markers KLK2: 0.1 µL semen and MSMB: 0.01 µL semen, and vaginal material marker CYP2B7P: 1 in 1000 dilution of a whole vaginal swab. The method successfully detected most body fluids in a range of simple mixtures with 77 out of 80 markers observed when expected.
      The developed one-step endpoint RT-PCR assays lack the sensitivity and precision required for forensic casework and provide little benefit when compared with standard two-step endpoint RT-PCR, other than minimal time and cost savings, similar sensitivity, and improved precision for some markers. As both methods utilise endpoint RT-PCR, they have the same narrow linear dynamic range. The novel method is therefore similarly susceptible to varied RNA input, a major disadvantage of this approach. The limited sensitivity and precision consistently encountered with endpoint RT-PCR - regardless of cDNA synthesis strategy - could be addressed by a real-time PCR approach.


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