Mock Forensic Samples Research Articles

An examination of differences in epigenetic methylation of saliva type samples based on collection method.

In the context of forensic casework, it is imperative to both establish a DNA profile from biological specimens and accurately identify the specific bodily fluid source. To achieve this, DNA methylation markers have been developed for the differentiation of blood, semen, vaginal epithelial secretions, and saliva samples. Saliva, alternatively referred to as oral fluid, is recognized for its heterogeneous cellular composition, characterized by a mixture of epithelial, leukocytic, and bacterial cells. Consequently, our research has revealed variations in methylation percentages that correlate with the method employed for collecting saliva samples. To investigate these concepts, we scrutinized four CpG markers situated within or in proximity to the BCAS4, SLC12A8, SOX2OT, and FAM43A genes. Subsequently, we designed primers based on bioinformatically transformed reference sequences for these markers and rigorously assessed their quality by examining dimer and hairpin formation, melting temperature, and specificity. These loci were identified as saliva markers based on either buccal swabs or spit collection. Yet, there has been minimal or no research conducted to explore the variations in methylation between different collection methods. For this study, buccal, lip, tongue, spit, and nasal swabs were collected from 20 individuals (N=100). Mock forensic samples, which include chewing gum (N=10) and cigarettes (N=10), were also tested. DNA was extracted, bisulfite converted, then amplified using in-house designed assays, and pyrosequenced. The methylation levels were compared to other body fluids (semen, blood, vaginal epithelia, and menstrual blood [N=32]). A total of 608 pyrosequencing results demonstrated that sampling location and collection method can greatly influence the level of methylation, highlighting the importance of examining multiple collection/deposition methods for body fluids when developing epigenetic markers.

(Un)Reliable detection of menstrual blood in forensic casework - evaluation of the Seratec® PMB test with mock samples.

The identification of the type of body fluid in crime scene evidence may be crucial, so that the efforts are high to reduce the complexity of these analyses and to minimize time and costs. Reliable immunochromatographic rapid tests for specific and sensitive identification of blood, saliva, urine and sperm secretions are already routinely used in forensic genetics. The recently introduced Seratec® PMB test is said to detect not only hemoglobin, but also differentiate menstrual blood from other secretions containing blood (cells) by detecting D-dimers. In our experimental set-up, menstrual blood could be reliably detected in mock forensic samples. Here, the result was independent of sample age and extraction buffer volume. It was also successfully demonstrated that all secretions without blood cells were negative for both, hemoglobin (P) and D-dimer (M). However, several blood cell-containing secretions/tissues comprising blood (injury), nasal blood, postmortem blood and wound crust also demonstrated positive results for D-dimer (M) and were therefore false positives. For blood (injury) and nasal blood, this result was reproduced for different extraction buffer volumes. The results of this study clearly demonstrate that the Seratec® PMB test is neither useful nor suitable for use in forensic genetics because of the great risk of false positive results which can lead to false conclusions, especially in sexual offense or violent acts.

Droplet-based optical trapping for cell separation in mock forensic samples.

Optical tweezers have a wide range of uses for mechanical manipulation of objects in the microscopic range. This includes both living and static cells in a variety of biomedical and research applications. Single-focus optical tweezers, formed by focusing a laser beam through a high numerical aperture immersion objective, create a significant force, which enables controlled transport of a variety of different cell types and morphologies in three dimensions. Optical tweezers have been previously reported to capture and separate spermatozoa from a reconstituted simulated postcoital sample. We report herein the development of a simplified, more efficient cell transfer protocol that can separate and isolate both spermatozoa as well as leukocytes, with similar efficiencies as those previously reported. The new cell transfer method was used to separate sperm cells from a reconstituted mixture of spermatozoa and vaginal epithelial cells, with complete STR profiles developed from 50 cells with little evidence of contribution from the female contributor to the mixture. This modified protocol was then used to separate 21 samples of enriched leukocytes, with trapped cells ranging from 5 to 22 cells. Complete STR profiles were developed from as few as 10 leukocytes. Thus, with minimal sample preparation and a short trapping time, this method has the potential to provide an alternative to traditional differential extraction methods for separation of sperm:nonsperm mixtures while also providing versatility for separation of cells with differing morphologies.

Rapid and visual detection of specific bacteria for saliva and vaginal fluid identification with the lateral flow dipstick strategy.

Identification of body fluids is critical for crime scene reconstruction, and a source of investigation source of investigative leads. In recent years, microbial DNA analysis using sequencing and quantitative real-time polymerase chain reaction have been used to identify body fluids. However, these techniques are time-consuming, expensive, and require complex workflows. In this study, a new method for simultaneous detection of Streptococcus salivarius and Lactobacillus crispatus using polymerase chain reaction (PCR) in combination with a lateral flow dipstick (LFD) was developed to identify saliva and vaginal fluid in forensic samples. LFD results can be observed with the naked eye within 3min with a sensitivity of 0.001ng/µL DNA. The PCR-LFD assay was successfully used to detect S. salivarius and L. crispatus in saliva and vaginal fluid respectively, and showed negative results in blood, semen, nasal fluid, and skin. Moreover, saliva and vaginal fluid were detectable even at an extremely high mixing ratio of sample DNA (1:999). Saliva and vaginal fluid were identified in various mock forensic samples. These results indicate that saliva and vaginal fluid can be effectively detected by identifying S. salivarius and L. crispatus, respectively. Furthermore, we have shown that DNA samples used to identify saliva and vaginal fluid can also provide a complete short tandem repeat (STR) profile when used as source material for forensic STR profiling. In summary, our results suggest that PCR-LFD is a promising assay for rapid, simple, reliable, and efficient identification of body fluids.

Validation of a novel fluorescent probe-based real-time PCR assay to detect saliva-specific unmethylated CpG sites for saliva identification

The identification of saliva from forensic samples is often important to establish what happened at a crime scene, especially in sexual assault cases. Recently, CpG sites that are specifically methylated or unmethylated in saliva have been reported as markers for saliva identification. In this study, we designed a fluorescent probe-based real-time polymerase chain reaction (PCR) assay for analyzing the methylation status of two neighboring CpG sites, which we previously found were saliva-specifically unmethylated. Specificity analysis using various types of body fluid/tissue samples demonstrated a probe detecting the unmethylation of the two CpG sites reacted only to saliva DNA, indicating this probe as an all-or-nothing marker for the presence of saliva DNA. Sensitivity analysis demonstrated that the detection limit was 0.5 ng saliva DNA as input for bisulfite conversion, while we confirmed a negative effect of larger amounts of non-saliva DNA on sensitivity in the analysis of saliva–vaginal DNA mixtures. We finally validated the applicability of this test to swabs from licked skin and bottles after drinking as mock forensic samples in comparison with other saliva-specific markers. We confirmed the potential usefulness of this test for skin samples, from which a saliva-specific mRNA was not detected reliably, while the ingredients in several beverages might affect methylation analysis. Given the simplicity of real-time PCR as well as the high specificity and sensitivity of the test, we believe the developed method is suitable for routine forensic analysis and can play an important role in saliva identification.

Application of DNA repair for Streptococcus salivarius DNA-based identification of saliva from ultraviolet-exposed samples

Forensic samples are commonly influenced by various environmental factors, including ultraviolet (UV) irradiation; thus, forensic applications of DNA repair (e.g., PreCR™, Restorase®) have been investigated, focusing on short tandem repeat typing. However, current DNA-based examinations are used for both human and body fluid identification. This study thus aims to clarify the efficacy of a DNA repair approach for Streptococcus salivarius DNA-based identification of saliva from UV-damaged samples. Artificial UV-damaged genomic DNA of S. salivarius, drop saliva stains, and buccal swabs were used to evaluate the effects of DNA repair on S. salivarius DNA detection by using PreCR™ repair reagent. To evaluate forensic applications, we prepared mock forensic samples by exposing them to environmental conditions. Melting curve analysis following real-time PCR was applied for qualitatively detecting S. salivarius DNA with a specific melting peak of 80.5°C±0.4°C (n=10, mean ± 3SD). Single PCR was used for quantitative and qualitative analyses, whereas dual PCR was used for S. salivarius DNA qualitative detection. DNA repair experiments using artificial UV-damaged samples revealed a significant increase of only the quantitative value of genomic DNA samples by DNA repair. Moreover, significant quantitative DNA repair effects were not observed in all mock forensic samples, indicating the limitations of DNA repair for actual cell-derived DNA samples. Whereas, differences of qualitative results (with or without detection) were generated for mock forensic samples; thus, we consider the DNA repair strategy as an additional approach for S. salivarius DNA-based identification of saliva from environmentally damaged evidence.

Taking the ‘Secret’ out of secretions: evaluation of the ParaDNA body fluid ID system at NSWHP FASS

ABSTRACTThe ParaDNA Body Fluid ID (BFID) system has been developed for use as either a screening or confirmatory test for the simultaneous identification of six biological fluids: saliva, vaginal fluid, seminal fluid, spermatozoa, peripheral blood and menstrual blood. The ParaDNA BFID kit consists of a closed, single-tube assay, incorporating multiplexed reverse transcription of mRNA and cDNA amplification, followed by fluorescent melt curve analysis with LGC’s proprietary HyBeacon DNA Probe technology. The ParaDNA instrument and early access version of the BFID kit were evaluated in this study. The evaluation included an assessment of the system’s useability, sensitivity, substrate specificity, and performance with mixtures and mock forensic samples. The ParaDNA BFID system was user-friendly, and capable of detecting most body fluids at neat concentrations. However, the system was unable to detect diluted fluids. The system’s capacity to detect saliva was also limited. Performance of the assay with different substrates was most influenced by the effectiveness of the ParaDNA sample collection tool. The tool sampled well from dried stains on fabrics, but was less effective at sampling from rayon swabs and other absorbent substrates. The system is capable of detecting fluids in mixtures, although detection appeared dependent on the combination of fluids tested.

Sperm cell purification from mock forensic swabs using SOMAmer™ affinity reagents

We have demonstrated a proof of concept with affinity-based purification of sperm cells from mock forensic samples using SOMAmer™ reagents, DNA-based affinity reagents developed by SomaLogic, Inc. SOMAmer reagents were selected in vitro using whole-cell SELEX to bind specifically with intact, detergent-treated sperm cells. Successful separation of sperm from epithelial cells and their debris was demonstrated using buccal swabs with added semen. Primarily male DNA profiles were generated from sperm cells eluted from the types of cotton swabs typically used for rape kit evidence collection. The quality of sperm DNA isolated from samples purified using SOMAmers is comparable to existing commercially available differential extraction-based methods at higher sperm concentrations. This purification method is simple, offers relatively rapid (<2hr) sperm purification, and can potentially be automated using robotic workstations. This work serves as proof of concept that demonstrates the first use of SOMAmer reagents as affinity ligands to bind sperm cells. With further development, this technique can potentially be used for high-throughput sexual assault forensic casework.

Simple and rapid identification of saliva by detection of oral streptococci using direct polymerase chain reaction combined with an immunochromatographic strip

In this paper, we describe the development of a novel method to detect oral bacteria by combining direct polymerase chain reaction (direct PCR) with an immunochromatographic strip (ICS), enabling the identification of saliva in forensic samples. Direct PCR was first used to directly amplify specific oral bacterial sequences (from Streptococcus sanguinis and Streptococcus salivarius) from swab samples, circumventing the need for tedious sample preparation steps such as cell lysis and DNA extraction and purification. The resultant amplicons were then colorimetrically detected on an ICS, a much more convenient, cost-effective, and user-friendly detection method than those currently available, thereby allowing the presence or absence of the target oral bacteria to be determined with the naked eye. Moreover, the entire analysis process was performed rapidly and with ease using this combination of direct PCR amplification from swab samples and ICS-based amplicon detection. This method successfully detected S. sanguinis and S. salivarius in most of the saliva swab samples tested, and returned negative results using blood, semen, urine, and vaginal fluid swab samples. Furthermore, S. sanguinis and S. salivarius were detected in a large number of mock forensic samples using this technique, which suggests that direct PCR and ICS-based detection of oral bacteria is sufficient to demonstrate the presence of saliva. Thus, we believe that the proposed method could be very useful for the identification of saliva in forensic applications.

Design, optimisation and preliminary validation of a human specific loop-mediated amplification assay for the rapid detection of human DNA at forensic crime scenes

The identification of samples at a crime scene which require forensic DNA typing has been the focus of recent research interest. We propose a simple, but sensitive analysis system which can be deployed at a crime scene to identify crime scene stains as human or non-human. The proposed system uses the isothermal amplification of DNA in a rapid assay format, which returns results in as little as 30min from sampling. The assay system runs on the Genie II device, a proven in-field detection system which could be deployed at a crime scene. The results presented here demonstrate that the system was sufficiently specific and sensitive and was able to detect the presence of human blood, semen and saliva on mock forensic samples.

Identification of canine saliva using mRNA-based assay.

A dog saliva analysis in addition to a bite-mark analysis may be important for evidence when a crime involves a dog bite. In this study, the utility of detecting canine saliva-specific mRNAs to identify canine saliva was evaluated. Canine saliva swabs (n = 20), urine swabs (n = 20), body surface swabs (n = 20), whole blood samples (n = 10), human saliva (n = 20), human skin surface swabs (n = 20), and human whole blood (n = 20) were tested. The saliva-specific genes encoding statherin (STATH), carbonic anhydrase VI (CA-VI), and dog allergens (Canf1 and Canf2) were analyzed as candidate genes. Moreover, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as confirmation of canine mRNA extraction. STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs were detected in 19/20, 1/20, 11/20, 4/20, and 20/20 saliva samples, respectively. The STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs did not exhibit cross-reactivity with samples of human origin. This mRNA-based assay was also able to detect canine saliva in mock forensic samples. The results of this study indicated that the detection of STATH mRNA is useful for the identification of canine saliva, and GAPDH is a suitable marker for canine mRNA extraction.

A novel method for sex determination by detecting the number of X chromosomes.

A novel method for sex determination, based on the detection of the number of X chromosomes, was established. Current methods, based on the detection of the Y chromosome, can directly identify an unknown sample as male, but female gender is determined indirectly, by not detecting the Y chromosome. Thus, a direct determination of female gender is important because the quality (e.g., fragmentation and amelogenin-Y null allele) of the Y chromosome DNA may lead to a false result. Thus, we developed a novel sex determination method by analyzing the number of X chromosomes using a copy number variation (CNV) detection technique (the comparative Ct method). In this study, we designed a primer set using the amelogenin-X gene without the CNV region as the target to determine the X chromosome copy number, to exclude the influence of the CNV region from the comparative Ct value. The number of X chromosomes was determined statistically using the CopyCaller software with real-time PCR. All DNA samples from participants (20 males, 20 females) were evaluated correctly using this method with 1-ng template DNA. A minimum of 0.2-ng template DNA was found to be necessary for accurate sex determination with this method. When using ultraviolet-irradiated template DNA, as mock forensic samples, the sex of the samples could not be determined by short tandem repeat (STR) analysis but was correctly determined using our method. Thus, we successfully developed a method of sex determination based on the number of X chromosomes. Our novel method will be useful in forensic practice for sex determination.

PCR Applications in Identification of Saliva Samples Exposed to Different Conditions (Streptococci Detection based)

Oral streptococci represent about 20% of the total oral bacteria, so if it is possible to detect the presence of oral specific bacteria from a forensic specimen by Polymerase chain reaction, this could be used to verify the presence of saliva. Aim of this study is detection of Streptococcus salivarius which is one of the most common streptococci in oral bacteria and Streptococcus mutans which is common in cases of dental caries in various body fluids and skin swabs and assessment of which one of both organisms is more reliable in saliva identification, cross sectional study on Egypt population. Negative control samples (15 samples) were taken from various body fluids (urine, semen) and skin swabs. Mock forensic samples (85 samples) included fresh saliva, saliva, cotton fabrics contaminated with saliva, cigarette butts, bitten apple and semen mixed with saliva samples). DNA extraction was done using DNeasy blood and tissue kit (Qiagen, Tokyo, Japan). Polymerase chain reaction was done for DNA amplification using Polymerase chain reaction master mix then gel electrophoresis was done for samples qualification. Control bacteria were S. salivarius and Streptococcus mutans. Streptococcus salivarius was detected in 83.5% of all saliva contained samples and S. mutans was detected in 67% of saliva contained samples. Both bacteria were not detected in other body fluids and skin swabs, so S. salivarius is more reliable in saliva identification as well as differentiating it from other body fluids. Polymerase chain reaction is valuable in detection of saliva by detecting S. salivarius.

Purifying and Concentrating Genomic DNA from Mock Forensic Samples Using Millipore Amicon Filters

Regenerated cellulose filters are used for concentrating and purifying genomic DNA from casework samples, due to the high yields and low retentate volumes that these filters provide. The Millipore Ultracel YM-100 is an example of this filter type and has been available to the forensics community for this application since 1990. In 2002, Millipore introduced the Amicon line of vertical filters that provide a larger area for filtration and have a dead space to prevent spinning to dryness. In the present study, Amicon filters were optimized in terms of g force and spin times for their ability to concentrate and purify genomic DNA. The Amicon Ultra 0.5 mL 30 K was used with mock forensic samples containing as little as 160 buccal cells, 20 nL of blood, or 8 nL of semen. In conclusion, the Amicon line of filters can be used to purify genomic DNA from small numbers of cells.

Rapid screening of synthetic cathinones as trace residues and in authentic seizures using a portable mass spectrometer equipped with desorption electrospray ionization

Recently, the surge in synthetic cathinone abuse has become a matter of public concern. With the influx of confiscated synthetic cathinones dramatically on the rise, laboratory workloads are expected to increase at a similar rate. This prompts the need for rapid analytical methods capable of detecting and identifying such compounds. A ruggedized, portable ion trap mass spectrometer capable of desorption electrospray ionization mass spectrometry (DESI-MS) was used to rapidly characterize various synthetic cathinones. Target analytes were directly analyzed as trace residues on various substrates and as major components in authentic, powder-based forensic evidence. Physical transfer swabs can also be examined with this method, allowing efficient screening of large areas and geometrically complex samples. Method validity was tested on trace residues, mock forensic samples, and authentic evidentiary seizures, yielding low- to sub-ng detection limits from several substrates of interest to crime scene investigation. Analyte confirmation was accomplished through MS(2) analysis, providing characteristic fragmentation similar to that reported in literature. High-throughput analysis was demonstrated with no significant instrumental carryover, even for powdered samples. The robustness of this DESI-MS method to multi-component samples was examined, marked by high chemical specificity. Coupling DESI-MS with portable instrumentation allowed sensitive and selective examination of synthetic cathinones from various substrates, in complex mixtures, and directly from mock and authentic forensic evidence. This instrumental method has the potential to assess the evidentiary value of forensic samples at crime scenes, reducing backlogs and expediting criminal investigations.

Activated Paper Surfaces for the Rapid Hybridization of DNA through Capillary Transport

The development of low-cost, accurate, and equipment-free diagnostic tests is crucial to many clinical, laboratory, and field applications, including forensics and medical diagnostics. Cellulose fiber-based paper is an inexpensive, biodegradable, and renewable resource, the use of which as a biomolecule detection matrix and support confers several advantages compared to traditional materials such as glass. In this context, a new, facile method for the preparation of surface functionalized papers bearing single-stranded probe DNA (ssDNA) for rapid target hybridization via capillary transport is presented. Optimized reaction conditions were developed that allowed the direct, one-step activation of standard laboratory filters by the inexpensive and readily available bifunctional linking reagent, 1,4-phenylenediisothiocyanate. Such papers were thus amenable to subsequent coupling of amine-labeled ssDNA under standard conditions widely used for glass-based supports. The intrinsic wicking ability of the paper matrix facilitated rapid sample elution through arrays of probe DNA, leading to significant, detectable hybridization in the time required for the sample liquid to transit the vertical length of the strip (less than 2 min). The broad applicability of these paper test strips as rapid and specific diagnostics in "real-life" situations was exemplified by the discrimination of amplicons generated from canine and human mitochondrial and genomic DNA in mock forensic samples.

Analysis of 17-ketosteroids in human urine by gas chromatography-mass spectrometry: conditioning of acid-hydrolysis of conjugate form and application of extractive-derivatization method

We have attempted to establish the analytical condition of 17-ketosteroids (17-KS), i.e., androsterone (An), etiocholanolone (Eti) and dehydroepiandrosterone (DHEA) for identification of human urine with gas chromatography-mass spectrometry. First, we optimized the condition of acid-hydrolysis of 17-KS conjugates in human urine. Heating at 100°C for 20 min with 3% of HCl gave maximum yields of liberated 17-KS, and excess HCl and heating for a long period caused a degradation of An and DHEA. Subsequently, we attempted to apply the heptafluorobutyryl (HFB) derivatization method using an extraction solvent containing HFB-chloride. This method is convenient because 17-KS are derivatized and extracted simultaneously at room temperature. The highest recoveries were obtained when n-hexane containing 5% HFB-chloride and 0.1% triethylamine was used as the extraction solvent. Using this method, we successfully detected the 17-KS in mock forensic samples. We therefore suggest that this method is useful for the identification of human urine in forensic science.

Evaluation of automatable silica-based extraction methods for low quantity samples

This study evaluated four commercial silica-based extraction chemistries using serum and mock forensic samples to determine which automatable method is best suited to isolate DNA from low quantity samples. Data from quantitative polymerase chain reaction assays identified the PrepFiler™ Forensic DNA Extraction kit as the superior extraction method with average yields equal to or better than those obtained with organic and other commercial silica-based methods tested.

A Simple Identification Method of Saliva by Detecting Streptococcus salivarius Using Loop‐Mediated Isothermal Amplification

We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop-mediated isothermal amplification (LAMP). The LAMP primer set was designed using S. salivarius-specific sequences of glucosyltransferase K. To simplify the procedure, the sample was prepared by boiling and mutanolysin treatment only, and the entire analytical process was completed within 2.5 h. The cut-off value was set at 0.1 absorbance units, measured at 660 nm, upon termination of the reaction. S. salivarius was identified in all saliva samples, but was not detected in other body fluids or on the skin surface. Using this method, S. salivarius was successfully detected in various mock forensic samples. We therefore suggest that this approach is useful for the identification of saliva in forensic practice.

Using oral microbial DNA analysis to identify expirated bloodspatter

Distinguishing expirated bloodstains (blood forced by airflow out of the nose, mouth or a chest wound) from impact spatter (blood from gunshots, explosives, blunt force trauma and/or machinery accidents) is an important challenge in forensic science. Streptococcal bacteria are only found in the human mouth and saliva. This study developed a polymerase chain reaction (PCR) method that detects DNA from these bacteria as a sensitive tool to detect the presence of saliva. The PCR method was very specific to human oral streptococci, with no PCR product being made from human DNA or DNA from other microbes that were tested. It was also very sensitive, detecting as little as 60 fg of target DNA. The PCR amplification gave product with 99 out of 100 saliva samples tested. PCR was not inhibited by the presence of blood and could detect target DNA in expirated bloodstains in a range of materials and for up to 92 days after deposit on cardboard or cotton fabric. In a blind trial, the PCR method was able to distinguish three mock forensic samples that contained expirated blood from four that did not. Our data show that bacteria present in the oral cavity can be detected in bloodstains that contain saliva and therefore can potentially be used as a marker in forensic work to distinguish mouth-expirated bloodstains from other types of bloodstains.