Top

Published in:

01-11-2020 | Original Article

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

Authors: Kuo-Lan Liu, Li-Chin Tsai, Yu-Chih Lin, Nu-En Huang, Lih-Jing Yang, Chih-Wen Su, James Chun-I Lee, Adrian Linacre, Hsing-Mei Hsieh

Published in: International Journal of Legal Medicine | Issue 6/2020

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.

Available only for authorised users

Malsom S, Flanagan N, McAlister C, Dixon L (2009) The prevalence of mixed DNA profiles in fingernail samples taken from couples who co-habit using autosomal and Y-STRs. Forensic Sci Int Genet 3(2):57–62PubMedCrossRef

An JH, Shin KJ, Yang WI, Lee HY (2012) Body fluid identification in forensics. BMB Rep 45(10):545–553PubMedCrossRef

Old J, Schweers BA, Boonlayangoor PW, Fischer B, Miller KW, Reich K (2012) Developmental validation of RSID™-semen: a lateral flow immunochromatographic strip test for the forensic detection of human semen. J Forensic Sci 57(2):489–499PubMedCrossRef

Allery JP, Telmon N, Mieusset R, Blanc A, Rougé D (2001) Cytological detection of spermatozoa: comparison of three staining methods. J Forensic Sci 46(2):349–351PubMedCrossRef

Henky H, Budiningsih Y, Widiatmaka W (2011) The validity of rapid test to detect prostate-specific antigen (PSA) in seminal fluid. Med J Indonesia 20(4):278–282CrossRef

Sato I, Sagi M, Ishiwari A, Nishijima H, Ito E, Mukai T (2002) Use of the “SMITEST” PSA card to identify the presence of prostate-specific antigen in semen and male urine. Forensic Sci Int 127(1–2):71–74PubMedCrossRef

Graves HC, Sensabaugh GF, Blake ET (1985) Postcoital detection of a male-specific semen protein: application to the investigation of rape. N Engl J Med 312(6):338–343PubMedCrossRef

Suttipasit P, Wongwittayapanich S (2018) Detection of prostate specific antigen and semenogelin in specimens from female rape victims. J Forensic Legal Med 54:102–108CrossRef

Casey DG, Domijan K, MacNeill S, Rizet D, O'connell D, Ryan J (2017) The persistence of sperm and the development of time since intercourse (TSI) guidelines in sexual assault cases at forensic science Ireland, Dublin, Ireland. J Forensic Sci 62(3):585–592PubMedCrossRef

10.

Hanson EK, Ballantyne J (2016) Enhanced DNA profiling of the semen donor in late reported sexual assaults: use of Y-chromosome-targeted pre-amplification and next generation Y-STR amplification systems. In: Forensic DNA Typing Protocols. Springer, pp 185–200

11.

Katilius E, Carmel AB, Koss H, O’Connell D, Smith BC, Sanders GM, LaBerge GS (2018) Sperm cell purification from mock forensic swabs using SOMAmer™ affinity reagents. Forensic Sci Int Genet 35:9–13PubMedCrossRef

12.

Costa S, Correia-de-Sá P, Porto MJ, Cainé L (2017) The use of laser microdissection in forensic sexual assault casework: pros and cons compared to standard methods. J Forensic Sci 62(4):998–1006PubMedCrossRef

13.

Muro CK, Doty KC, de Souza Fernandes L, Lednev IK (2016) Forensic body fluid identification and differentiation by Raman spectroscopy. Forensic Chem 1:31–38CrossRef

14.

Roeder AD, Haas C (2013) mRNA profiling using a minimum of five mRNA markers per body fluid and a novel scoring method for body fluid identification. Int J Legal Med 127(4):707–721PubMedCrossRef

15.

Ingold S, Dørum G, Hanson E, Berti A, Branicki W, Brito P, Elsmore P, Gettings K, Giangasparo F, Gross T (2018) Body fluid identification using a targeted mRNA massively parallel sequencing approach–results of a EUROFORGEN/EDNAP collaborative exercise. Forensic Sci Int Genet 34:105–115PubMedCrossRef

16.

Sirker M, Fimmers R, Schneider P, Gomes I (2017) Evaluating the forensic application of 19 target microRNAs as biomarkers in body fluid and tissue identification. Forensic Sci Int Genet 27:41–49PubMedCrossRef

17.

Frumkin D, Wasserstrom A, Budowle B, Davidson A (2011) DNA methylation-based forensic tissue identification. Forensic Sci Int Genet 5(5):517–524PubMedCrossRef

18.

Jung SE, Cho S, Antunes J, Gomes I, Uchimoto ML, Oh YN, Di Giacomo L, Schneider PM, Park MS, van der Meer D (2016) A collaborative exercise on DNA methylation based body fluid typing. Electrophoresis 37(21):2759–2766PubMedCrossRef

19.

Jones PA (2012) Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 13(7):484–492PubMedCrossRef

20.

Hashimshony T, Zhang J, Keshet I, Bustin M, Cedar H (2003) The role of DNA methylation in setting up chromatin structure during development. Nat Genet 34(2):187–192PubMedCrossRef

21.

Miranda TB, Jones PA (2007) DNA methylation: the nuts and bolts of repression. J Cell Physiol 213(2):384–390PubMedCrossRef

22.

Song F, Smith JF, Kimura MT, Morrow AD, Matsuyama T, Nagase H, Held WA (2005) Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression. Proc Natl Acad Sci U S A 102(9):3336–3341PubMedPubMedCentralCrossRef

23.

Wasserstrom A, Frumkin D, Davidson A, Shpitzen M, Herman Y, Gafny R (2013) Demonstration of DSI-semen—a novel DNA methylation-based forensic semen identification assay. Forensic Sci Int Genet 7(1):136–142PubMedCrossRef

24.

Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual:9.14–19.19. Cold Spring Harbor Laboratory Press, New York

25.

Yu-Chih L, Li-Chin T, James Chun-I L, Kuo-Lan L, Jason Tze-Cheng T, Adrian L, Hsing-Mei H (2016) Novel identification of biofluids using a multiplex methylation-specific PCR combined with single-base extension system. Forensic Sci Med Pathol 12(2):128–138CrossRef

26.

Balamurugan K, Bombardi R, Duncan G, McCord B (2014) Identification of spermatozoa by tissue-specific differential DNA methylation using bisulfite modification and pyrosequencing. Electrophoresis 35(21–22):3079–3086PubMedCrossRef

27.

Tanaka K, Okamoto A (2007) Degradation of DNA by bisulfite treatment. Bioorg Med Chem Lett 17(7):1912–1915PubMedCrossRef

28.

Yu-Chih L, Li-Chin T, James Chun-I L, Chih-Wen S, Jason Tze-Cheng T, Adrian L, Hsing-Mei H (2016) Novel identification of biofluids using a multiplex methylation sensitive restriction enzyme-PCR system. Forensic Sci Int Genet 25:157–165CrossRef

29.

Krausz C, Sandoval J, Sayols S, Chianese C, Giachini C, Heyn H, Esteller M (2012) Novel insights into DNA methylation features in spermatozoa: stability and peculiarities. PLoS One 7(10):e44479PubMedPubMedCentralCrossRef

30.

Reinius LE, Acevedo N, Joerink M, Pershagen G, Dahlén SE, Greco D, Söderhäll C, Scheynius A, Kere J (2012) Differential DNA methylation in purified human blood cells: implications for cell lineage and studies on disease susceptibility. PLoS One 7(7):e41361PubMedPubMedCentralCrossRef

31.

Farkas SA, Milutin-Gašperov N, Grce M, Nilsson TK (2013) Genome-wide DNA methylation assay reveals novel candidate biomarker genes in cervical cancer. Epigenetics 8(11):1213–1225PubMedCrossRef

32.

Souren NY, Lutsik P, Gasparoni G, Tierling S, Gries J, Riemenschneider M, Fryns J-P, Derom C, Zeegers MP, Walter J (2013) Adult monozygotic twins discordant for intra-uterine growth have indistinguishable genome-wide DNA methylation profiles. Genome Biol 14(5):R44PubMedPubMedCentralCrossRef

33.

Slieker RC, Bos SD, Goeman JJ, Bovée JV, Talens RP, van der Breggen R, Suchiman HED, Lameijer E-W, Putter H, van den Akker EB (2013) Identification and systematic annotation of tissue-specific differentially methylated regions using the Illumina 450k array. Epigenetics Chromatin 6(1):26PubMedPubMedCentralCrossRef

34.

Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3--new capabilities and interfaces. Nucleic Acids Res 40(15):e115. https://doi.org/10.1093/nar/gks596PubMedPubMedCentralCrossRef

35.

Koressaar T, Remm M (2007) Enhancements and modifications of primer design program Primer3. Bioinformatics 23(10):1289–1291PubMedCrossRef

36.

Yu H, Diamandis EP (1995) Prostate-specific antigen in milk of lactating women. Clin Chem 41(1):54–58PubMedCrossRef

37.

Lövgren J, Valtonen-André C, Marsal K, Liua H, Lundwall Å (1999) Measurement of prostate-specific antigen and human glandular kallikrein 2 in different body fluids. J Androl 20(3):348–355PubMed

38.

Cox M (1991) A study of the sensitivity and specificity of four presumptive tests for blood. J Forensic Sci 36(5):1503–1511PubMedCrossRef

39.

Kaye S (1949) Acid phosphatase test for identification of seminal stains. J Lab Clin Med 34(5):728–732PubMed

40.

Vernarecci S, Ottaviani E, Agostino A, Mei E, Calandro L, Montagna P (2015) Quantifiler® Trio Kit and forensic samples management: a matter of degradation. Forensic Sci Int Genet 16:77–85PubMedCrossRef

41.

Sidstedt M, Hedman J, Romsos EL, Waitara L, Wadsö L, Steffen CR, Vallone PM, Rådström P (2018) Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR. Anal Bioanal Chem 410(10):2569–2583PubMedPubMedCentralCrossRef

Title: Identification of spermatozoa using a novel 3-plex MSRE-PCR assay for forensic examination of sexual assaults
Authors: Kuo-Lan Liu
Li-Chin Tsai
Yu-Chih Lin
Nu-En Huang
Lih-Jing Yang
Chih-Wen Su
James Chun-I Lee
Adrian Linacre
Hsing-Mei Hsieh
Publication date: 01-11-2020
Publisher: Springer Berlin Heidelberg
Published in: International Journal of Legal Medicine / Issue 6/2020
Print ISSN: 0937-9827
Electronic ISSN: 1437-1596
DOI: https://doi.org/10.1007/s00414-020-02285-1

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

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

Abstract

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Abstract

Please log in to get access to this content

Other articles of this Issue 6/2020

Deadly injuries through recoilless anti-tank weapons while military shooting practice—two case studies from Germany and Switzerland

German population data for 18 X-STRs: a hexaplex PCR adding two clusters of X-STRs to the Argus X-12 set and expanding the German haplotype databases

Evaluation and review of ways to differentiate sources of ethanol in postmortem blood

Haplotype analysis of 36 Y-STR loci in a Chinese Han population from Anhui Province, Eastern China

Aspiration in lethal drug abuse—a consequence of opioid intoxication

Cerebellar heterotopia of infancy in sudden infant death syndrome: an observational neuropathological study of four cases