Body Fluid Identification Research Articles

Developmental validation of an mRNA kit: A 5-dye multiplex assay designed for body-fluid identification

Identifying the sources of biosamples found at crime scenes is crucial for forensic investigations. Among the markers used for body fluid identification (BFI), mRNA has emerged as a well-studied marker because of its high specificity and remarkable stability. Despite this potential, commercially available mRNA kits specifically designed for BFI are lacking. Therefore, we developed an mRNA kit that includes 21 specific mRNA markers of body fluids, along with three housekeeping genes for BFI, to identify four forensic-relevant fluids (blood, semen, saliva, and vaginal fluids). In this study, we tested 451 single-body-fluid samples, validated the universality of the mRNA kit, and obtained a gene expression profile. We performed the validation studies in triplicates and determined the sensitivity, specificity, stability, precision, and repeatability of the mRNA kit. The sensitivity of the kit was found to be 0.1 ng. Our validation process involved the examination of 59 RNA mixtures, 60 body fluids mixtures, and 20 casework samples, which further established the reliability of the kit. Furthermore, we constructed five classifiers that can handle single-body fluids and mixtures using this kit. The classifiers output possibility values and identify the specific body fluids of interest. Our results showed the reliability and suitability of the BFI kit, and the Random Forest classifier performed the best, with 94% precision. In conclusion, we developed an mRNA kit for BFI which can be a promising tool for forensic practice.

Comparing endpoint and real-time PCR for forensic body fluid identification

ABSTRACT Identifying the body fluid source of stains can give an indication of activities, prioritize samples for DNA profiling, and provide probative evidence when the presence of DNA alone does not. Messenger RNA profiling assays using endpoint reverse-transcription polymerase-chain reaction (RT-PCR), were developed to address the issues with conventional body fluid identification and have since been used in forensic casework. However, endpoint RT-PCR has a narrow linear dynamic range (LDR), and there is currently no specific method for mRNA quantification to direct case strategy and optimize input template. Real-time reverse-transcription quantitative PCR (RT-qPCR) collects data during the amplification phase which reflects the starting quantity. The application of RT-qPCR assays as a quantification precursor to endpoint RT-PCR was investigated using previously described assays for circulatory blood, saliva, menstrual fluid, spermatozoa, seminal fluid, and vaginal material. While blood marker SLC4A1 and vaginal marker CYP2B7P showed a strong relationship, the remaining body fluid markers lacked a robust predictive relationship, likely due to differences in LDR. CYP2B7P and SLC4A1 showed high sensitivity when compared with other markers – resulting in a greater number of data points for estimation of the linear relationship. Compared with endpoint, the RT-qPCR assays had higher precision, LDR, and sensitivity.

Assessment of Blood and Semen Detection and DNA Collection from Swabs up to Three Months after Deposition on Five Different Cloth Materials.

Body fluid identification plays a crucial role in criminal investigations. Because of their presence in many cases, blood and semen are the most relevant body fluids in forensic sciences. Based on antigen-antibody reactions binding unique proteins for each body fluid, serological assays represent one of the most rapid and highly specific tests for blood and semen. Currently, few studies have assessed the factors affecting body fluid identification by applying these assays. This work aimed to study the effect of different fabrics from clothes and time since deposition on identification through immunochromatographic tests for blood and semen, DNA isolation, and STR profiling from these samples. Body fluids were deposited on black- and white-dyed denim and cotton fabrics, and on leather. Afterward, blood and semen were sampled at 1 day, 30 days, and 90 days after deposition and identified by using the SERATEC® HemDirect Hemoglobin Test and the PSA Semiquant and SERATEC® BLOOD CS and SEMEN CS tests, respectively. Laboratory and crime scene tests presented similar performances for the detection of blood and semen stains on every tested fabric. No differences were found on band intensities between timepoints for all fabrics. It was possible to recover and identify blood and semen samples up to three months after deposition and to obtain full STR profiles from all the tested fabrics. Both body fluid STR profiles showed differences in their quality between 1 and 90 days after deposition for all fabrics except for black cotton for semen samples. Future research will expand the results, assessing body fluid identification on other substrates and under different environmental conditions.

Advances in body fluid identification: MiRNA markers as powerful tool.

Body fluids are one of the most encountered types of evidence in any crime and are commonly used for identifying a person's identity. In addition to these, they are also useful in ascertaining the nature of crime by determining the ty pe of fluid such as blood, semen, saliva, urine etc. Body fluids collected from crime scenes are mostly found in degraded, trace amounts and/or mixed with other fluids. However, the existing immunological and enzyme-based methods used for differentiating these fluids show limited specificity and sensitivity in such cases. To overcome these challenges, a new method utilizing microRNA expression of the body fluids has been proposed. This method is believed to be non-destructive as well as sensitive in nature and researches have shown promising results for highly degraded samples as well. This systematic review focuses on and explores the use and reliability of miRNAs in body fluid identification. It also summarizes the researches conducted on various aspects of miRNA in terms of body fluid examination in forensic investigations.

Body fluids should be identified before estimating the time since deposition (TsD) in microbiome-based stain analyses for forensics.

Identification and the time since deposition (TsD) estimation of body fluid stains from a crime scene could provide valuable information for solving the cases and are always difficult for forensics. Microbial characteristics were considered as a promising biomarker to address the issues. However, changes in the microbiota may damage the specific characteristics of body fluids. Correspondingly, incorrect body fluid identification may result in inaccurate TsD estimation. The mutual influence is not well understood and limited the codetection. In the current study, saliva, semen, vaginal secretion, and menstrual blood samples were exposed to indoor conditions and collected at eight time points (from fresh to 30 days). High-throughput sequencing based on the 16S rRNA gene was performed to characterize the microbial communities. The results showed that a longer TsD could decrease the discrimination of different body fluid stains. However, the accuracies of identification still reached a quite high value even without knowing the TsD. Correspondingly, the mean absolute error (MAE) of TsD estimation significantly increased without distinguishing the types of body fluids. The predictive TsD of menstrual blood reached a quite low MAE (1.54 ± 0.39 d). In comparison, those of saliva (6.57 ± 1.17 d), semen (6.48 ± 1.33 d), and vaginal secretion (5.35 ± 1.11 d) needed to be further improved. The great effect of individual differences on these stains limited the TsD estimation accuracy. Overall, microbial characteristics allow for codetection of body fluid identification and TsD estimation, and body fluids should be identified before estimating TsD in microbiome-based stain analyses.IMPORTANCEEmerged evidences suggest microbial characteristics could be considered a promising tool for identification and time since deposition (TsD) estimation of body fluid stains. However, the two issues should be studied together due to a potential mutual influence. The current study provides the first evidence to understand the mutual influence and determines an optimal process for codetection of identification and TsD estimation for unknown stains for forensics. In addition, we involved aged stains into our study for identification of body fluid stains, rather than only using fresh stains like previous studies. This increased the predictive accuracy. We have preliminary verified that individual differences in microbiotas limited the predictive accuracy of TsD estimation for saliva, semen, and vaginal secretion. Microbial characteristics could provide an accurate TsD estimation for menstrual blood. Our study benefits the comprehensive understanding of microbiome-based stain analyses as an essential addition to previous studies.

Making the Most of Lateral Flow Immunochromatographic Tests: An Efficient Protocol to Recover DNA.

Lateral flow immunochromatographic (LFI) tests are widely used in both biomedical and forensic sciences for different applications. In forensic sciences, their main use is to detect body fluids at crime scenes. However, there are situations in which the amount of potential biological evidence is so low that DNA extraction is favored with respect to the identification of body fluids. Here, an efficient and quick protocol is presented to integrate the detection of body fluids through LFI with DNA extraction from a sample swab and buffer, providing a complete characterization of the biological evidence. This protocol is a modification of a general DNA extraction silica-based kit, whose main application is for blood and tissues. Thus, it could be carried out in different settings (forensic labs, hospitals, other testing labs) without the necessity of buying a specific kit for swabs. The validation of this protocol is supported by the results presented here and previous publications from our group, obtaining DNA in good quantity and with good quality. This proves the potential application of the protocol in both forensic scenarios, to fully characterize biological evidence, and biomedical settings, to molecularly confirm the results of LFI tests.

Identification of urine samples obtained in forensic cases by fourier transform infrared spectroscopy

Forensic sciences are a multidisciplinary science that covers all branches of science for justice. Forensic genetics is one of the sub-branches of forensic sciences. It is a branch of science that contributes to establishing a connection between crime and criminal by using all kinds of biological evidence (blood, semen, saliva, urine, vaginal fluid, hair, hair, sweat, saliva, etc.) and reaching the perpetrator with the genetic information obtained. Serological and immunological tests are used in the identification of body fluids found as evidence at the crime scene. The number of serological tests used in the past and today is limited; not all body fluids can be analyzed routinely. Many of the existing tests are not directly specific to the body fluid but are intended to identify intermediate metabolites. One such example is the urine sample. In the identification of urine, the presence of creatinine and amines in the urine is often used. In addition, these tests require the expenditure of separate consumables to identify each body fluid. For this reason, with the development of technology, in recent years, the use of spectroscopic methods that provide cost-free and easy analysis in the identification of body fluids has increased. Fourier Transform Infrared (FTIR) spectroscopy is one of the spectroscopic methods used to identify body fluids from biological samples found at the crime scene without loss of quantity and without damaging the sample, quickly and without loss of consumables. This study investigated the identification of urine stains obtained from the crime scene with FTIR spectroscopy without using serological materials. Functional groups, which we can express as identification regions, were determined as 13 different distinguishing points in the relevant molecules and their usability in forensic serological analyses was determined.

Phenotypic profiling based on body fluid traces discovered at the scene of crime: Raman spectroscopy of urine stains for race differentiation.

Modern criminal investigations heavily rely on trace bodily fluid evidence as a rich source of DNA. DNA profiling of such evidence can result in the identification of an individual if a matching DNA profile is available. Alternatively, phenotypic profiling based on the analysis of body fluid traces can significantly narrow down the pool of suspects in a criminal investigation. Urine stain is a frequently encountered specimen at the scene of crime. Raman spectroscopy offers great potential as a universal confirmatory method for the identification of all main body fluids, including urine. In this proof-of-concept study, Raman spectroscopy combined with advanced statistics was used for race differentiation based on the analysis of urine stains. Specifically, a Random Forest (RF) model was built, which allowed for differentiating Caucasian (CA) and African American (AA) descent donors with 90% accuracy based on Raman spectra of dried urine samples. Raman spectra were collected from samples of 28 donors varying in age and sex. This novel technology offers great potential as a universal forensic tool for phenotypic profiling of a potential suspect immediately at the scene of a crime, providing invaluable information for a criminal investigation.

Assessment of body fluid identification and DNA profiling after exposure to tropical weather conditions.

Despite current advances in body fluid identification, there are few studies evaluating the effect of environmental conditions. The present work assessed the detection of body fluids, blood, semen, and saliva, through lateral flow immunochromatographic (LFI) tests, exposed to tropical weather conditions over time, also evaluating the possibility of obtaining STR (short tandem repeat) profiles and identifying mitochondrial DNA (mtDNA) polymorphisms. Blood, semen, saliva samples, and mixtures of these fluids were deposited on polyester clothes and exposed to open-air tropical weather conditions for 1 month. The test versions from LFI (SERATEC®, Germany) Lab and crime scene (CS) used for the detection - one per each body fluid type - demonstrated that it is possible to identify body fluids and their mixtures up to 14 days after deposition. At 30 days, blood and semen were detected but not saliva. Full STR profiles were obtained from 14-day-old blood samples, and partial profiles were obtained from the remaining samples. It was possible to sequence mtDNA in the samples previously analyzed for STR profiling, and haplogroups could be assigned. In conclusion, this study demonstrated for the first time the possibility of body fluid identification and DNA profiling after exposure to tropical weather conditions for 1 month and also demonstrated the value of mtDNA analysis for compromised biological evidence.

An mRNA profiling assay incorporating coding region InDels for body fluid identification and the inference of the donor in mixed samples

Biological traces discovered at crime scenes hold significant significance in forensic investigations. In cases involving mixed body fluid stains, the evidentiary value of DNA profiles depends on the type of body fluid from which the DNA was obtained. Recently, coding region polymorphism analysis has proved to be a promising method for directly linking specific body fluids to their respective DNA contributors in mixtures, which may help to avoid "association fallacy" between separate DNA and RNA evidence. In this study, we present an update on previously reported coding region Single Nucleotide Polymorphisms (cSNPs) by exploring the potential application of coding region Insertion/Deletion polymorphisms (cInDels). Nine promising cInDels, selected from 70 mRNA markers based on stringent screening criteria, were integrated into an existing mRNA profiling assay. Subsequently, the body fluid specificity of our cInDel assay and the genotyping consistency between complementary DNA (cDNA) and genomic DNA (gDNA) were examined. Our study demonstrates that cInDels can function as important multifunctional genetic markers, as they provide not only the ability to confirm the presence of forensically relevant body fluids, but also the ability to associate/dissociate specific body fluids with particular donors.

Analysis of the fluorescent properties of vaginal fluid upon ageing

Detection and identification of body fluids are crucial aspects of forensic investigations, aiding in crime scene reconstructions and providing important leads. Although many methods have been developed for these purposes, no method is currently in use in the forensic field that allows rapid, non-contact detection and identification of vaginal fluids directly at the crime scene. The development of such technique is mainly challenged by the complex chemistry of the constituents, which can differ between donors and exhibits changes based on woman’s menstrual cycle. The use of fluorescence spectroscopy has shown promise in this area for other biological fluids. Therefore, the aim of this study was to identify specific fluorescent signatures of vaginal fluid with fluorescence spectroscopy to allow on-site identification. Additionally, the fluorescent properties were monitored over time to gain insight in the temporal changes of the fluorescent spectra of vaginal fluid. The samples were excited at wavelengths ranging from 200 to 600 nm and the induced fluorescence emission was measured from 220 to 700 nm. Excitation and emission maps (EEMs) were constructed for eight donors at seven time points after donation. Four distinctive fluorescence peaks could be identified in the EEMs, indicating the presence of proteins, fluorescent oxidation products (FOX), and an unidentified component as the dominant contributors to the fluorescence. To further asses the fluorescence characteristics of vaginal fluid, the fluorescent signatures of protein and FOX were used to monitor protein and lipid oxidation reactions over time. The results of this study provide insights into the intrinsic fluorescent properties of vaginal fluid over time which could be used for the development of a detection and identification method for vaginal fluids. Furthermore, the observed changes in fluorescence signatures over time could be utilized to establish an accurate ageing model.

Application of SNaPshot Technology in Semen-Specific cSNP Genetic Marker.

To explore the feasibility of genetic marker detection of semen-specific coding region single nucleotide polymorphism (cSNP) based on SNaPshot technology in semen stains and mixed body fluid identification. Genomic DNA (gDNA) and total RNA were extracted from 16 semen stains and 11 mixtures composed of semen and venous blood, and the total RNA was reverse transcribed into complementary DNA (cDNA). The cSNP genetic markers were screened on the validated semen-specific mRNA coding genes. The cSNP multiplex detection system based on SNaPshot technology was established, and samples were genotyped by capillary electrophoresis (CE). A multiplex detection system containing 5 semen-specific cSNPs was successfully established. In 16 semen samples, except the cSNP located in the TGM4 gene showed allele loss in cDNA detection results, the gDNA and cDNA typing results of other cSNPs were highly consistent. When detecting semen-venous blood mixtures, the results of cSNP typing detected were consistent with the genotype of semen donor and were not interfered by the genotype of venous blood donor. The method of semen-specific cSNPs detection by SNaPshot technology method can be applied to the genotyping of semen (stains) and provide information for determining the origin of semen in mixed body fluids (stains).

Expanding the scope of methylation-sensitive restriction enzyme (MSRE) PCR for forensic identification of body fluids through the novel use of methylation-dependent restriction enzymes (MDRE) and the combination of autosomal and Y-chromosomal markers

Methylation-sensitive/-dependent restriction enzyme (MSRE/MDRE) PCR can be performed to detect hypomethylated or hypermethylated CpG sites. With the combined use of different tissue-specific CpG markers, MSRE/MDRE-PCR leads to tissue-specific methylation patterns (TSMPs), enabling the correlation of DNA samples to their source tissue. MSRE/MDRE assays can use the same platform as forensic STR typing and offer many advantages in the field of forensic body fluid detection. In the present study, we aimed to establish MSRE assays for the detection of blood, saliva, vaginal secretion, and semen, using markers from literature and from our own database search. We designed two different MSRE test-sets, which include two novel Y-chromosomal non-semen markers, and enable differentiation between female and male non-semen samples. Furthermore, we established an MSRE/MDRE semen approach, which includes only Y-chromosomal non-semen and semen markers. This Y-semen multiplex PCR utilizes the novel combination of the methylation-sensitive enzyme SmaI and the methylation-dependent enzyme GlaI, which enables more sensitive detection of male body fluids within male/female DNA mixtures. Our validation tests confirmed that MSRE/MDRE assays exhibit high sensitivity, similar to that of STR typing.

Stand‐off Raman spectroscopy is a promising approach for the detection and identification of bloodstains for forensic purposes

AbstractStand‐off Raman spectroscopy combined with chemometrics was demonstrated to have great potential for detecting and identifying biological stains from a distance for forensic purposes. Bloodstain spectra were collected using a handheld Raman spectrometer equipped with a stand‐off attachment and compared with spectra acquired with a benchtop Raman microscope. The two sets of spectra were visually similar, supporting the potential use of a handheld Raman spectrometer with stand‐off attachment for blood detection at the scene of crime. In addition, the stand‐off Raman spectra of bloodstains were correctly identified using a previously built SVM‐DA model for the identification of all main body fluids (Muro et al., Forensic Chemistry, 2016). Stand‐off Raman spectroscopy opens a new potential opportunity for further development of Raman spectroscopy as a universal tool for detecting and identifying body fluid traces for forensic purposes.

Partial validation of multiplexed real-time quantitative PCR assays for forensic body fluid identification

Confirmatory body fluid identification using messenger RNA (mRNA) is a well-established technique to address issues encountered with conventional testing – such as poor sensitivity, specificity, and a lack of available tests for all body fluids of interest. For over a decade, endpoint reverse-transcription polymerase chain reaction (RT-PCR) assays have been used in forensic casework for such purposes. However, in comparison with real-time quantitative RT-PCR (RT-qPCR), endpoint RT-PCR has lower sensitivity, precision, and linear dynamic range.This research details the multiplexing and partial validation of confirmatory RT-qPCR assays. We have previously described novel assays for a range of body fluid targets and identified an optimal commercial kit for their amplification. Here, multiplexing was undertaken to form three assays: circulatory blood (SLC4A1) and menstrual fluid (STC1), saliva (HTN3) and vaginal material (CYP2B7P), and spermatozoa (PRM1) and seminal fluid (KLK2), all including a synthetic internal control RNA.Partial validation of the multiplexed assays incorporated the MIQE guidelines, ISO requirements, and SWGDAM guidelines. Using receiver operating characteristic (ROC) curves, each marker was significantly different from an uninformative assay and optimal cut-offs were all above 35 cycles. All assays showed a wide LDR (ranging from 3 to 5 logs with most R2 > 0.99), and high precision (most mean CV < 1 %). STC1 showed some instances of sporadic expression in blood, semen, and vaginal material at high CT values. CYP2B7P showed off-target expression in semen and blood. The sensitivities were approximated as; saliva: 1 in 1,000 dilution of a whole buccal swab, circulatory blood: 0.01–0.1 µL blood, menstrual fluid: 1 in 10,000 dilution of a whole menstrual swab, spermatozoa: 0.001 µL semen, seminal fluid: 0.01 µL semen, and vaginal material: 1 in 1,000 dilution of a whole vaginal swab.A total of 16 mock body fluid extract mixtures and 18 swab mixtures were tested and had 100% and 99% detection of target markers below each specific cut-off, respectively. Some mixtures containing high volumes of blood and semen showed off-target CYP2B7P expression. The successful application of a probabilistic model to the RT-qPCR data was also demonstrated. Further work will involve full developmental validation.

A combined molecular approach utilizing microbial DNA and microRNAs in a qPCR multiplex for the classification of five forensically relevant body fluids.

Body fluid identification is an essential step in the forensic biology workflow that can assist DNA analysts in determining where to collect DNA evidence. Current presumptive tests lack the specificity that molecular techniques can achieve; therefore, molecular methods, including microRNA (miRNA) and microbial signature characterization, have been extensively researched in the forensic community. Limitations of each method suggest combining molecular markers to increase the discrimination efficiency of multiple body fluids from a single assay. While microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have shown promise in fluids with low microbial abundance (blood and semen). This project synergized the benefits of microRNAs and microbial DNA to identify multiple body fluids using DNA extracts. A reverse transcription (RT)-qPCR duplex targeting miR-891a and let-7g was validated, and miR-891a differential expression was significantly different between blood and semen. The miRNA duplex was incorporated into a previously reported qPCR multiplex targeting 16S rRNA genes of Lactobacillus crispatus, Bacteroides uniformis, and Streptococcus salivarius to presumptively identify vaginal/menstrual secretions, feces, and saliva, respectively. The combined classification regression tree model resulted in the presumptive classification of five body fluids with 94.6% overall accuracy, now including blood and semen identification. These results provide proof of concept that microRNAs and microbial DNA can classify multiple body fluids simultaneously at the quantification step of the current forensic DNA workflow.

Research progress and potential application of microRNA and other non-coding RNAs in forensic medicine.

At present, epigenetic markers have been extensively studied in various fields and have a high value in forensic medicine due to their unique mode of inheritance, which does not involve DNA sequence alterations. As an epigenetic phenomenon that plays an important role in gene expression, non-coding RNAs (ncRNAs) act as key factors mediating gene silencing, participating in cell division, and regulating immune response and other important biological processes. With the development of molecular biology, genetics, bioinformatics, and next-generation sequencing (NGS) technology, ncRNAs such as microRNA (miRNA), circular RNA (circRNA), long non-coding RNA (lncRNA), and P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) are increasingly been shown to have potential in the practice of forensic medicine. NcRNAs, mainly miRNA, may provide new strategies and methods for the identification of tissues and body fluids, cause-of-death analysis, time-related estimation, age estimation, and the identification of monozygotic twins. In this review, we describe the research progress and application status of ncRNAs, mainly miRNA, and other ncRNAs such as circRNA, lncRNA, and piRNA, in forensic practice, including the identification of tissues and body fluids, cause-of-death analysis, time-related estimation, age estimation, and the identification of monozygotic twins. The close links between ncRNAs and forensic medicine are presented, and their research values and application prospects in forensic medicine are also discussed.

Identification of Mixtures of Two Types of Body Fluids Using the Multiplex Methylation System and Random Forest Models.

Body fluid mixtures are complex biological samples that frequently occur in crime scenes, and can provide important clues for criminal case analysis. DNA methylation assay has been applied in the identification of human body fluids, and has exhibited excellent performance in predicting single-source body fluids. The present study aims to develop a methylation SNaPshot multiplex system for body fluid identification, and accurately predict the mixture samples. In addition, the value of DNA methylation in the prediction of body fluid mixtures was further explored. In the present study, 420 samples of body fluid mixtures and 250 samples of single body fluids were tested using an optimized multiplex methylation system. Each kind of body fluid sample presented the specific methylation profiles of the 10 markers. Significant differences in methylation levels were observed between the mixtures and single body fluids. For all kinds of mixtures, the Spearman's correlation analysis revealed a significantly strong correlation between the methylation levels and component proportions (1:20, 1:10, 1:5, 1:1, 5:1, 10:1 and 20:1). Two random forest classification models were trained for the prediction of mixture types and the prediction of the mixture proportion of 2 components, based on the methylation levels of 10 markers. For the mixture prediction, Model-1 presented outstanding prediction accuracy, which reached up to 99.3% in 427 training samples, and had a remarkable accuracy of 100% in 243 independent test samples. For the mixture proportion prediction, Model-2 demonstrated an excellent accuracy of 98.8% in 252 training samples, and 98.2% in 168 independent test samples. The total prediction accuracy reached 99.3% for body fluid mixtures and 98.6% for the mixture proportions. These results indicate the excellent capability and powerful value of the multiplex methylation system in the identification of forensic body fluid mixtures.

Development of a multiplex reverse transcription qPCR assay for identification of saliva, blood, and semen

ABSTRACT The identification of body fluids can provide important information for case processing in forensic investigations. Accurate identification through messenger RNA (mRNA) is effective, especially in cases of sexual assault, where body fluids from one or more contributors are mixed. In this study, a saliva, blood, and semen simultaneous identification reverse transcription-quantitative polymerase chain reaction (SBS RT-qPCR) assay using mRNA from a DNA/RNA co-extraction method was developed. The body fluid-specific mRNA markers histatin 3 (HTN3), haemoglobin subunit beta (HBB), and protamine 1 (PRM1) were selected for identification of saliva, blood, and sperm cells, respectively. The assay accurately identified saliva, blood, and semen without cross-reaction with 10 body fluids. The limits of detection (LoD) were 1/102-, 1/105-, and 1/103-fold dilutions of total RNA in saliva, blood, and semen, respectively. Furthermore, the assay accurately identified trace amounts of body fluids in mixed samples at a 58:1:1 ratio. Finally, it was possible to analyse the origin of body fluids by obtaining an STR profile using the DNA/RNA co-extraction method. The SBS RT-qPCR assay was able to simultaneously identify each body fluid with efficiency in sample consumption. Therefore, it is advisable to use the DNA/RNA co-extraction method to identify mixed body fluids in sexual assault cases.

The role of DNA in forensic science: A comprehensive review

Forensic genetics, leveraging molecular tools and scientific applications, has witnessed significant advancements in DNA analysis over the last three decades. These progressions have enhanced the discrimination power, speed, and sensitivity of DNA profiling methods, enabling the analysis of challenging samples. This article explores the significance of forensic genetics in criminal investigations, traces the historical evolution of DNA analysis techniques, and presents recent developments in the field. The article aims to provide a comprehensive understanding of the crucial role of forensic genetics in criminal investigations and sheds light on the latest trends and breakthroughs in this area. The evolution of DNA typing from ABO blood typing to the current standard of short tandem repeat (STR) analysis is discussed, along with alternative DNA analysis methods, such as Y-chromosome analysis and single nucleotide polymorphism (SNP) typing. Massively parallel sequencing (MPS) represents a groundbreaking advancement, enabling whole genome sequencing and addressing complex cases. The article also covers recent innovations, including DNA methylation analysis, body fluid identification, forensic DNA phenotyping, and genetic genealogy, highlighting their potential benefits in forensic investigations. Despite these advancements, standard STR profiling remains the gold standard due to its established protocols and databases. Ethical considerations regarding data privacy and cost implications are crucial as these technologies continue to progress in their pursuit of justice.