A simple identification method for vaginal secretions using relative quantification of Lactobacillus DNA.

In criminal investigations there are some cases in which identifying the presence of vaginal secretions provides crucial evidence in proving sexual assault. However, there are no methods for definitively identifying vaginal secretions. In the present study, we focused on Lactobacillus levels in vaginal secretions and developed a novel identification method for vaginal secretions by relative quantification based on real time PCR. We designed a Lactobacillus conserved region primer pair (LCP) by aligning 16S rRNA gene sequences from major vaginal Lactobacillus species (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii), and selected the human specific primer pair (HSP) as an endogenous control for relative quantification. As a result, the ΔCt (ΔCt=Ct[LCP]-Ct[HSP]) values of vaginal secretions (11 out of 12 samples) were significantly lower than those of saliva, semen and skin surface samples, and it was possible to discriminate between vaginal secretions and other body fluids. For the one remaining sample, it was confirmed that the predominant species in the microflora was not of the Lactobacillus genus. The ΔCt values in this study were calculated when the total DNA input used from the vaginal secretions was 10pg or more. Additionally, the ΔCt values of samples up to 6-months-old, which were kept at room temperature, remained unchanged. Thus, we concluded in this study that the simple ΔCt method by real time PCR is a useful tool for detecting the presence of vaginal secretions.

[1]  Woo Ick Yang,et al.  Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA , 2013, International Journal of Legal Medicine.

[2]  J. Sobel,et al.  Emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora. , 1990, Reviews of infectious diseases.

[3]  P. Walsh,et al.  A rapid chemiluminescent method for quantitation of human DNA. , 1992, Nucleic acids research.

[4]  Jack Ballantyne,et al.  mRNA Profiling for Body Fluid Identification by Multiplex Quantitative RT‐PCR * , 2007, Journal of forensic sciences.

[5]  James M Robertson,et al.  Evaluation of mRNA marker specificity for the identification of five human body fluids by capillary electrophoresis. , 2012, Forensic science international. Genetics.

[6]  吉田 日南子,et al.  法生物学的試料由来の DNA に対する定量法の検証 , 2007 .

[7]  W Parson,et al.  mRNA profiling for the identification of blood--results of a collaborative EDNAP exercise. , 2011, Forensic science international. Genetics.

[8]  L. Ripani,et al.  Molecular identification of vaginal fluid by microbial signature. , 2012, Forensic science international. Genetics.

[9]  E. Gharehbaghi-Schnell,et al.  Messenger RNA profiling: a novel method for body fluid identification by real-time PCR. , 2006, Forensic science international.

[10]  Marcus J Claesson,et al.  The genus Lactobacillus--a genomic basis for understanding its diversity. , 2007, FEMS microbiology letters.

[11]  J. Menten,et al.  Quantification of bacterial species of the vaginal microbiome in different groups of women, using nucleic acid amplification tests , 2012, BMC Microbiology.

[12]  J. Schwebke,et al.  Correlation of behaviors with microbiological changes in vaginal flora. , 1999, The Journal of infectious diseases.

[13]  W Parson,et al.  RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise. , 2012, Forensic science international. Genetics.

[14]  Ursel M. E. Schütte,et al.  The vaginal bacterial communities of Japanese women resemble those of women in other racial groups. , 2010, FEMS immunology and medical microbiology.

[15]  T. Kaku,et al.  Defensins in saliva and the salivary glands , 2003, Medical Electron Microscopy.

[16]  H. Shojo,et al.  Identification of feces by detection of Bacteroides genes. , 2013, Forensic science international. Genetics.

[17]  H. Iwase,et al.  Detection of bacterial 16S ribosomal RNA genes for forensic identification of vaginal fluid. , 2012, Legal medicine.

[18]  R. Fleming,et al.  The use of bacteria for the identification of vaginal secretions. , 2010, Forensic science international. Genetics.

[19]  Xavier Messeguer,et al.  DnaSP, DNA polymorphism analyses by the coalescent and other methods , 2003, Bioinform..

[20]  M. Nadkarni,et al.  Quantitative Analysis of Diverse Lactobacillus Species Present in Advanced Dental Caries , 2004, Journal of Clinical Microbiology.

[21]  Jong-Lyul Park,et al.  Genome-wide mRNA profiling and multiplex quantitative RT-PCR for forensic body fluid identification. , 2013, Forensic science international. Genetics.

[22]  J. Jakubowska,et al.  mRNA Profiling for Vaginal Fluid and Menstrual Blood Identification. , 2016, Methods in molecular biology.

[23]  Mohammed R. Islam,et al.  Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods. , 2004, Microbiology.

[24]  Kazuyuki Saito,et al.  A novel method for the identification of saliva by detecting oral streptococci using PCR. , 2009, Forensic science international.

[25]  A. Meier,et al.  Influence of the normal menstrual cycle on vaginal tissue, discharge, and microflora. , 2000, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[26]  W Parson,et al.  RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: results of a fourth and fifth collaborative EDNAP exercise. , 2014, Forensic science international. Genetics.

[27]  P. Toselli,et al.  Expression of Membrane-associated Mucins MUC1 and MUC4 in Major Human Salivary Glands , 2002, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[28]  Li Tao,et al.  Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences , 2002, Journal of applied microbiology.

[29]  J. Ballantyne,et al.  Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. , 2003, Forensic science international.

[30]  H. M. Bramley,et al.  Normal vaginal flora. , 1979, Genitourinary medicine.

[31]  Y. Koga,et al.  Suppressive effect of Lactobacillus gasseri OLL 2716 (LG21) on Helicobacter pylori infection in humans. , 2001, The Journal of antimicrobial chemotherapy.

[32]  中園 毅彦.  Successful DNA typing of urine stains using a DNA purification kit following dialfiltration , 2006 .

[33]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[34]  Miss A.O. Penney.  (b) , 1974, The New Yale Book of Quotations.

[35]  J. Dhar,et al.  What is normal vaginal flora? , 1997, British medical journal.

[36]  Titia Sijen,et al.  Vaginal microbial flora analysis by next generation sequencing and microarrays; can microbes indicate vaginal origin in a forensic context? , 2012, International Journal of Legal Medicine.

[37]  C. Haas,et al.  mRNA profiling for body fluid identification , 2008 .

[38]  Erin K Hanson,et al.  Highly specific mRNA biomarkers for the identification of vaginal secretions in sexual assault investigations. , 2013, Science & justice : journal of the Forensic Science Society.

[39]  H. Curtis.  What is normal vaginal flora? , 1997, Genitourinary medicine.

[40]  S. Witkin,et al.  Bacterial flora of the female genital tract: function and immune regulation. , 2007, Best practice & research. Clinical obstetrics & gynaecology.

[41]  Á. Carracedo,et al.  RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise. , 2013, Forensic science international. Genetics.