Research Article| Volume 59, 102712, July 2022

Evaluation and simultaneous determination of rectal mucosa markers by multiplex reverse transcription-PCR for biological evidence of sexual assault with anal penetration


      • Sexual assault with anal penetration is closely related to child or male victims.
      • Few procedures use biological samples to identify anal penetration.
      • The identification of rectal mucosa might help to prove such a sexual assault.
      • Rectal mucosa mRNA markers were screened and a multiplex RT-PCR was developed.
      • Precise determination of rectal mucosa could be performed with this procedure.


      Sexual assault with anal penetration is closely related to child sexual abuse or male victims. However, it is difficult to prove such an act by using biological samples collected from the surface of a suspected object because procedures for identifying rectal mucosa have not been developed sufficiently. Therefore, for the specific identification of rectal mucosa, mRNA markers reported to be characteristically expressed in the rectum were screened and a multiplex RT-PCR procedure was developed for the simultaneous determination of those candidate markers. The detectability and specificity of rectal mucosa candidate markers were evaluated using rectal mucosa samples and forensically relevant body fluids. Diluted or mixed samples were also tested to evaluate the applicability of this procedure for forensic casework. As a result, simultaneous amplification and determination of the selected candidates (PHGR1, MUC13, CLCA1, MEP1A, CDX1, and ZG16) and reference gene were successfully performed using a multiplex RT-PCR assay combined with capillary electrophoresis and fragment analysis. Applying the cutoff values, none of the other body fluids cross-reacted with rectal mucosa candidate markers. Because the low sensitivity and detectability of some candidate markers could be compensated for by their simultaneous detection, all six candidate markers were considered to be applicable as rectal mucosa markers. Besides, the developed assay should not be performed on suspicious fecal samples directly because these markers could be positive in the fecal samples themselves. The developed multiplex RT-PCR procedure might not be suitable for minute or diluted samples; however, it might be resistant to contamination with sexual assault-related body fluids. In conclusion, the simultaneous determination of selected rectal mucosa markers with a biological sample collected from the surface of a suspected object could be beneficial for criminal investigation of sexual assault with anal penetration.


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