International Journal of Forensic Sciences

Citation: Liang W, et al. Research Progress in Forensic Body Fluids Identification Based on Nucleic Acid Molecules. Int J Forens Sci 2016, 1(3): 000114.

Research Progress in Forensic Body Fluids Identification Based on Nucleic Acid Molecules

LiangW*, Li Z and Zhang L
Department of Forensic Genetics, West China School of Basic Science and Forensic Medicine, Sichuan University, China
Mini Review
Volume 1 Issue 3 - 2016
Received Date: September 18, 2016
Published Date: October 18, 2016

*Corresponding author: LiangW, Department of Forensic Genetics, West China School of Basic Science and Forensic Medicine, Sichuan University, China, Email:

Full Text

Identifying the type and origin of body fluids left at a crime scene is helpful to crime scene reconstruction for it can link the sample donors and actual criminal acts [1]. As Hanson et al. [2] pointed out, the identification of the biological material might be crucial to the prosecution of the case. The appearance of a person’s DNA only suggests that he once has been to the crime scene, but his acts were unknown for us. If we can identify origin of the biological material, for example semen stains, we can get more information from the crime scene. Numerous types of body fluid identification methods have been developed since last century, such as chemical tests [3-5], immunological tests [6,7], protein catalytic activity tests [8,9], spectroscopic methods [10-15] and microscopy [16]. However, these conventional body fluid identification methods are prone to various limitations such as sample consumption, intensive labor, time consumption, varying degrees of sensitivity and specificity [17,18]. Recent developments in nucleic acids detection methods have expanded the available molecules for forensic body fluid identification. Detection of DNA methylation [19-24], body fluid-specific microbial DNA [25-28], mRNA profiling [29-34] and expression profiling of miRNA [2,35-40] are the typically new methods of body fluid identification based on nucleic acids. DNA methylation is one of the most important epigenome makers [41]. Recent studies indicated that DNA possessed tissue-specific methylation patterns and there are several chromosone segments called tissue-specific differently methylated regions (tDMRs) which show varying methylation patterns according to tissue or cell type [41]. Comparing with other makers, simple extraction and purification methods, high sensivity and specificity make DNA methylation a more favourable detection method [19,21,24,42]. According to previous studies, only semen had specific DNA methylation loci that USP49 and DACT1 is unmethylated in 90 percent semen samples but high methylated in all blood, saliva, vaginal secretion and menstrual blood samples [43]. The degree of DNA methylation is affected by many various exogenous and endogenous factors such as ageing [44-46], nutrition and diets [47,48], early life experience [49,50], exposure to pollutants as well as social environments [51], which restrict the use of DNA methylation in body fluids identification.

Various bacteria reside in human body. Different body regions contain different kinds of bacteria. The use of bacteria for the identification of body fluids has been widely investigated, with Streptococcus species being used for the identification of saliva. Lactobacilli species have been found to be the predominant bacteria in the vagina of women. Only these two body fluids possessed relatively stable microflora. Similar to DNA methylation, several factors influenced the specificity and sensitivity of the bacteria [52-54]. Different age groups may have different bacteria in saliva and vaginal secretion, for instance, the diversity of bacteria of saliva in children and old man was more abundant than adults. Besides, antibiotics may change major species of bacteria in body fluids [55]. MRNA (message RNA) showed best performance in body fluid identification among the new markers. European DNA profiling group (EDNAP) has intensively studied and evaluated mRNA profiling of different body fluids and obtained satisfactory results in their collaborative exercises [56-60]. MiRNAs are one of small, non-coding RNA molecules with a length of 18-25 nucleotides [61-64]. The intrinsically small size and tissue-specific expression pattern make it less prone to aggressive environmental factors comparing against mRNA [17,18]. Another advantage of miRNA is in mixture stains, while mRNA profiling identifies origin of the sample, miRNA profiling is helpful to further distinguish the major donor and minor donor. Quantitative detection of mRNAs and miRNAs were performed in tissues with different RIN values (3-10) simultaneously in a study [65]. The expression of miRNAs was quite stable while the Ct values of mRNAs rose gradually as the RIN values decreased, which suggest miRNAs were indeed more stable comparing with mRNAs. The expression of some miRNA was more abundant than other body fluids which can differentiate target body fluid from others. Several studies have screened miRNA for body fluid identification [2,35-40], however the overlap in body fluididentification miRNA markers between studies is low. The agreement on miRNAs for body fluid identification should be reached. Combination of mRNAs and miRNAs may achieve better results.

  1. An JH, Shin KJ, Yang WI, Lee HY (2012) Body fluid identification in forensics. BMB Rep 45: 545-553.
  2. Hanson EK, Lubenow H, Ballantyne J (2009). Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs. Anal Biochem 387: 303-314.
  3. Barni F, Lewis SW, Berti A, Miskelly GM, Lago G (2007) Forensic application of the luminol reaction as a presumptive test for latent blood detection. Talanta 72: 896-913.
  4. Castelló A, Alvarez M, Verdú F (2002) Accuracy, Reliability, and Safety of Luminol in Bloodstain Investigation. Canadian Society of Forensic Science Journal 35: 113-121.
  5. Blum LJ, Esperan ÇAP, Rocquefelte S (2006) A New High-Performance Reagent and Procedure for Latent Bloodstain Detection Based on Luminol Chemiluminescence. Canadian Society of Forensic Science Journal 39: 81-99.
  6. Healy DA, Hayes CJ, Leonard P, McKenna L, O'Kennedy R (2007) Biosensor developments: application to prostate-specific antigen detection. Trends Biotechnol 25: 125-131.
  7. Virkler K, Lednev IK (2009) Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Sci Int 188: 1-17.
  8. Hedman J, Gustavsson K, Ansell R (2008) Using the new Phadebas® Forensic Press test to find crime scene saliva stains suitable for DNA analysis. Forensic Science International: Genetics Supplement Series 1: 430-432.
  9. Myers JR, Adkins WK (2008) Comparison of modern techniques for saliva screening. J Forensic Sci 53: 862-867.
  10. Claybourn M, Ansell M (2000) Using Raman Spectroscopy to solve crime: inks, questioned documents and fraud. Sci Justice 40: 261-271.
  11. Day JS, Edwards HG, Dobrowski SA, Voice AM (2004) The detection of drugs of abuse in fingerprints using Raman spectroscopy II: cyanoacrylate-fumed fingerprints. Spectrochim Acta A Mol Biomol Spectrosc 60: 1725-1730.
  12. Day JS, Edwards HGM, Dobrowski SA, Voice AM (2004) The detection of drugs of abuse in fingerprints using Raman spectroscopy I: latent fingerprints. Spectrochim Acta A Mol Biomol Spectrosc 60: 563- 568.
  13. Mazzella WD, Buzzini P (2005) Raman spectroscopy of blue gel pen inks. Forensic Sci Int 152: 241-247.
  14. Pang BC, Cheung BK (2007) Identification of human semenogelin in membrane strip test as an alternative method for the detection of semen. Forensic Sci Int 169: 27-31.
  15. Sturgeon CM, Ellis AR (2007) Improving the comparability of immunoassays for prostate-specific antigen (PSA): progress and problems. Clinica chimica acta 381: 85-92.
  16. Romero-Montoya L, Martínez-Rodríguez H, Pérez MA, Argüello-García R (2011) Relationship of spermatoscopy, prostatic acid phosphatase activity and prostate-specific antigen (p30) assays with further DNA typing in forensic samples from rape cases. Forensic Sci Int 206: 111-118.
  17. Setzer M, Juusola J, Ballantyne J (2008) Recovery and stability of RNA in vaginal swabs and blood, semen, and saliva stains. J Forensic Sci 53: 296-305.
  18. Zubakov D, Kokshoorn M, Kloosterman A, Kayser M (2009) New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16-year-old stains. Int J Legal Med 123: 71-74.
  19. Frumkin D, Wasserstrom A, Budowle B, Davidson A (2011) DNA methylation-based forensic tissue identification. Forensic Sci Int Genet 5: 517-524.
  20. Lee HY, Park MJ, Choi A, An JH, Yang WI, et al. (2012) Potential forensic application of DNA methylation profiling to body fluid identification. Int J Legal Med 126: 55-62.
  21. Madi T, Balamurugan K, Bombardi R, Duncan G, McCord B (2012) The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing. Electrophoresis 33: 1736-1745.
  22. An JH, Choi A, Shin KJ, Yang WI, Lee HY (2013) DNA methylation-specific multiplex assays for body fluid identification. Int J Legal Med 127: 35-43.
  23. Wasserstrom A, Frumkin D, Davidson A, Shpitzen M, Herman Y, et al. (2013) Demonstration of DSI-semen- -A novel DNA methylation-based forensic semen identification assay. Forensic Sci Int Genet 7: 136- 142.
  24. Choi A, Shin KJ, Yang WI, Lee HY (2014) Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA. Int J Legal Med 128: 33-41.
  25. Akutsu T, Motani H, Watanabe K, Iwase H, Sakurada K (2012) Detection of bacterial 16S ribosomal RNA genes for forensic identification of vaginal fluid. Leg Med (Tokyo) 14(3): 160-162.
  26. Benschop CC, Quaak FC, Boon ME, Sijen T, Kuiper I (2012) Vaginal microbial flora analysis by next generation sequencing and microarrays; can microbes indicate vaginal origin in a forensic context? Int J Legal Med 126:303-310.
  27. Giampaoli S, Berti A, Valeriani F, Gianfranceschi G, Piccolella A, et al. (2012) Molecular identification of vaginal fluid by microbial signature. Forensic Sci Int Genet 6(5): 559-564.
  28. Hsu L, Power D, Upritchard J, Burton J, Friedlander R, et al. (2012) Amplification of oral streptococcal DNA from human incisors and bite marks. Curr Microbiol 65(2): 207-211.
  29. Juusola J, Ballantyne J (2003) Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. Forensic Sci Int 135(2): 85-96.
  30. Juusola J, Ballantyne J (2005) Multiplex mRNA profiling for the identification of body fluids. Forensic Sci Int 152(1): 1-12.
  31. Nussbaumer C, Gharehbaghi-Schnell E, Korschineck I (2006) Messenger RNA profiling: a novel method for body fluid identification by real-time PCR. Forensic Sci Int 157(2-3): 181-186.
  32. Zubakov D, Hanekamp E, Kokshoorn M, van Ijcken W, Kayser M (2008) Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples. Int J Legal Med 122(2): 135-142.
  33. Sakurada K, Akutsu T, Watanabe K, Fujinami Y, Yoshino M (2011) Expression of statherin mRNA and protein in nasal and vaginal secretions. Leg Med (Tokyo) 13(6):309-313.
  34. Lindenbergh A, de Pagter M, Ramdayal G, Visser M, Zubakov D, et al. (2012) A multiplex (m)RNAprofiling system for the forensic identification of body fluids and contact traces. Forensic Sci Int Genet 6(5): 565-577.
  35. Zubakov D, Boersma AW, Choi Y, van Kuijk PF, Wiemer EA, et al. (2010) MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation. Int J Legal Med 124(3): 217-226.
  36. Courts C, Madea B (2011) Specific micro-RNA signatures for the detection of saliva and blood in forensic body-fluid identification. J Forensic Sci 56(6): 1464-1470.
  37. Hanson EK, Rekab K, Ballantyne J (2013) Binary logistic regression models enable miRNA profiling to provide accurate identification of forensically relevant body fluids and tissues. Forensic Science International: Genetics Supplement Series 4(1): e127- e128.
  38. Wang Z, Zhang J, Luo H, Ye Y, Yan J (2013) Screening and confirmation of microRNA markers for forensic body fluid identification. Forensic Sci Int Genet 7(1): 116-123.
  39. Park JL, Park SM, Kwon OH, Lee HC, Kim JY, et al. (2014) Microarray screening and qRT-PCR evaluation of microRNA markers for forensic body fluid identification. Electrophoresis 35(21-22): 3062-3068.
  40. Sauer E, Reinke AK, Courts C (2016) Differentiation of five body fluids from forensic samples by expression analysis of four microRNAs using quantitative PCR. Forensic Sci Int Genet 22: 89-99.
  41. Kader F, Ghai M (2015) DNA methylation and application in forensic sciences. Forensic Sci Int 249: 255-265.
  42. Heyn H, Moran S, Hernando-Herraez I, Sayols S, Gomez A, et al. (2013) DNA methylation contributes to natural human variation. Genome research 23: 1363-1372.
  43. Illingworth R, Kerr A, Desousa D, Jørgensen H, Ellis P, et al. (2008) A Novel CpG Island Set Identifies TissueSpecific Methylation at Developmental Gene Loci. PLoS Biol 6(1): e22.
  44. Gao ZH, Suppola S, Liu J, Heikkilä P, Jänne J, et al. (2002) Association of H19 Promoter Methylation with the Expression of H19 and IGF-II Genes in Adrenocortical Tumors. J Clin Endocrinol Metab 87(3): 1170-1176.
  45. Fraser HB, Lam LL, Neumann SM, Kobor MS (2012) Population-specificity of human DNA methylation. Genome Biol 13(2): 383-388.
  46. Day K, Waite LL, Thalacker-Mercer A, West A, Bamman MM (2013) Differential DNA methylation with age displays both common and dynamic features across human tissues that are influenced by CpG landscape. Genome Biol 14: 571-579.
  47. Hardy TM, Tollefsbol TO (2011) Epigenetic diet: impact on the epigenome and cancer. Epigenomics 3(4): 503-518.
  48. Park LK, Friso S, Choi SW (2012) Nutritional influences on epigenetics and age-related disease. Proc Nutr Soc 71: 75-83.
  49. Terry MB, Ferris JS, Pilsner R, Flom JD, Tehranifar P, et al. (2008) Genomic DNA methylation among women in a multiethnic New York City birth cohort. Cancer Epidemiol Biomarkers Prev 17: 2306-2310.
  50. Lam LL, Emberly E, Fraser HB, Neumann SM, Chen E, et al. (2012) Factors underlying variable DNA methylation in a human community cohort. Proc Natl Acad Sci USA 109(2): 17253-17260.
  51. Christensen BC, Houseman EA, Marsit CJ, Zheng S, Wrensch MR, et al. (2009) Aging and environmental exposures alter tissue-specific DNA methylation dependent upon CpG island context. PLoS genetics 5: e1000602.
  52. Vasquez A, Jakobsson T, Ahrne S, Forsum U, Molin G (2002) Vaginal Lactobacillus Flora of Healthy Swedish Women. J Clin Microbiol 40(8): 2746-2749.
  53. De Backer E, Verhelst R, Verstraelen H, Alqumber MA, Burton JP, et al. (2007) Quantitative determination by real-time PCR of four vaginal Lactobacillus species, Gardnerella vaginalis and Atopobium vaginae indicates an inverse relationship between L. gasseri and L. iners. BMC microbiology 7: 115.
  54. Witkin SS, Linhares IM, Giraldo P (2007) Bacterial flora of the female genital tract: function and immune regulation. Best Pract Res Clin Obstet Gynaecol 21(3): 347-354.
  55. Kang JG, Kim SH, Ahn TY (2006) Bacterial Diversity in the Human Saliva from Different Ages. J Microbiol 44(5): 572-576.
  56. Haas C, Hanson E, Anjos MJ, Ballantyne KN, Banemann R, et al. (2014) RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: results of a fourth and fifth collaborative EDNAP exercise. Forensic science international Genetics 8(1): 203-212.
  57. Haas C, Hanson E, Bar W, Banemann R, Bento AM, et al. (2011) mRNA profiling for the identification of blood--results of a collaborative EDNAP exercise. Forensic Sci Int Genet 5(1): 21-26.
  58. Haas C, Hanson E, Anjos MJ, Bär W, Banemann R, et al. (2012) RNA/DNA co-analysis from blood stains-- results of a second collaborative EDNAP exercise. Forensic Sci Int Genet 6(1): 70-80.
  59. Haas C, Hanson E, Anjos MJ, Banemann R, Berti A, et al. (2013) RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise. Forensic Sci Int Genet 7(2): 230-239.
  60. Haas C, Hanson E, Banemann R, Bento AM, Berti A, et al. (2015) RNA/DNA co-analysis from human skin and contact traces--results of a sixth collaborative EDNAP exercise. Forensic Sci Int Genet 16:139-147.
  61. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2): 281-297.
  62. Kim VN (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6(5): 376- 385.
  63. Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10(2): 126- 139.
  64. Courts C, Madea B (2010) Micro-RNA - A potential for forensic science? Forensic Sci Int 203: 106-111.
  65. Jung M, Schaefer A, Steiner I, Kempkensteffen C, Stephan C, et al. (2010) Robust microRNA stability in degraded RNA preparations from human tissue and cell samples. Clin Chem 56(6): 998-1006.

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