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Identification of spermatozoa using a novel 3-plex MSRE-PCR assay for forensic examination of sexual assaults

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Abstract

Identification of semen and then spermatozoa is essential to verify that sexual activity has occurred in alleged cases of sexual assault. Microscopic examination commonly used for spermatozoa identification is however time-consuming and can often lead to false-negative results for samples with deformed and, or, limited number of spermatozoa. To address this limitation, we report on a novel 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) assay to specifically identify spermatozoa. This assay is comprised of 3 markers: a digestive control marker (DC), sperm-specific marker (SP), and Y chromosome marker (SRY). A total of 214 samples from 10 body fluids or tissues were analyzed. Specificity testing showed that all the normal semen samples were unambiguously identified as being sperm-positive, and no other body fluid (or tissues) showed a sperm-specific signal in the electropherogram. Testing for sensitivity showed that 0.1 ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa by this assay. Mixture analyses illustrated the sensitivity of the assay when the vaginal/semen DNA ratio (80/0.1) was under 800 or the menstrual blood/semen DNA ratio (5/0.1) was under 50, the trace amounts (approximately 0.1 ng) of DNA from semen can still be identified by this 3-plex MSRE-PCR assay. This assay was also applied to the identification of 31 non-probative forensic samples from 18 sexual assault cases. The case studies showed that the 3-plex MSRE-PCR assay was an improvement in the sensitivity of spermatozoa detection.

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Funding

This study was supported by the Ministry of Science and Technology (NSC 102–2628-B-015-001-MY2), and Ministry of the Interior (108–0805-05-17-01) in Taiwan.

Author information

Authors and Affiliations

  1. Department of Forensic Science, Central Police University, 56 Shu-Jen Road, Kwei-San, Taoyuan, 33304, Taiwan, Republic of China

    Kuo-Lan Liu, Li-Chin Tsai & Hsing-Mei Hsieh

  2. Forensic Examination Division, Criminal Investigation Bureau, National Police Agency, 5 Lane 553, Chung Hsiao East Road Section 4, Xinyi District, Taipei, 11072, Taiwan, Republic of China

    Kuo-Lan Liu

  3. Taichung City Government Police Department, 588 Wenxin Road Section 2, Xitun District, Taichung City, 40758, Taiwan, Republic of China

    Yu-Chih Lin

  4. Forensic Biology Division, Criminal Investigation Bureau, National Police Agency, 5 Lane 553, Chung Hsiao East Road Section 4, Xinyi District, Taipei, 11072, Taiwan, Republic of China

    Nu-En Huang, Lih-Jing Yang & Chih-Wen Su

  5. Department of Forensic Medicine, College of Medicine, National Taiwan University, 1 Jen-Ai Road Section 1, Taipei, 10051, Taiwan, Republic of China

    James Chun-I Lee

  6. College of Science & Engineering, Flinders University, Adelaide, 5001, Australia

    Adrian Linacre

Authors
  1. Kuo-Lan Liu
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Contributions

All the authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Kuo-Lan Liu, Li-Chin Tsai, Yu-Chih Lin, Nu-En Huang, Lih-Jing Yang, and Chih-Wen Su. The first draft of the manuscript was written by James Chun-I Lee, Adrian Linacre, and Hsing-Mei Hsieh, and all the authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Hsing-Mei Hsieh.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

In this study, samples were collected after informed consent and following the procedures approved by the Institutional Review Board (IRB) of Taoyuan General Hospitals (IRB No. TYGH102011) and Antai-Tian-Sheng Memorial Hospital (IRB No. 18–074-B) in Taiwan. Neither the authors are affiliated to these hospitals nor the study was carried out in these hospitals. And the studies have been performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

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Informed consent was obtained from all individual participants included in the study.

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Liu, KL., Tsai, LC., Lin, YC. et al. Identification of spermatozoa using a novel 3-plex MSRE-PCR assay for forensic examination of sexual assaults. Int J Legal Med 134, 1991–2004 (2020). https://doi.org/10.1007/s00414-020-02285-1

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  • DOI: https://doi.org/10.1007/s00414-020-02285-1

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