A novel co-amplification system for simultaneous amplification of 23 Y-STR and identification of spermatozoa.

In alleged sexual assault cases, identification of the presence of spermatozoa at the crime scene, or on items of eventual significance, or associated with the body of the victim, is integral to the forensic investigation to support or refute the proposition that sexual act has occurred. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) system has been developed previously to identify spermatozoa based on the presence or absence of DNA methylation. This assay showed that 0.1 ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa even when there was excessively more DNA isolated from vaginal fluid than DNA from a semen extract (80 ng/0.1 ng) or a mix of the menstrual blood/semen DNA (5 ng/0.1 ng). In this study, we combine spermatozoa detection with co-amplification of 23 Y-STR loci. We perform standard validation steps to present a novel test that saves time and uses the same sample for both DNA typing and spermatozoa detection in the same reaction. The combined assay can identify Y-STR and spermatozoa simultaneously using just 0.1 ng semen DNA, even in the presence of 5 ng of DNA from a female (male/female:1/50). No other body fluid tested, such as saliva, gave a result for the presence of spermatozoa. A total of 9 non-probative forensic samples from 7 sexual assault cases were tested by this co-amplification system. In all cases, the same sperm-positive data were obtained, concordant with our previous study analyzed by only 3-plex MSRE-PCR, and the Y-STR results were also consistent with that analyzed by only PowerPlex® Y23 kit. The co-amplification will be beneficial for the limited samples in many criminal cases.

Spermatozoa identification by the 3-plex MSRE-PCR assay: a collaborative exercise.

Identification of semen and spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. In cases of alleged sexual assault where there is a long time gap between the incident and sample collection, or in cases of low sperm count, current methods have limitations of specificity, in the case of presumptive tests for semen, or the problem of recording spermatozoa by microscopy if they are few in number. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) assay using a spermatozoa-specific DNA methylated marker to identify spermatozoa has been reported previously by our laboratory. A key advantage over current methods is the increased sensitivity and specificity. A transition from a research tool to operational use requires blind trial testing and inter-laboratory trials. We report on a collaborative exercise where reagents of the 3-plex MSRE-PCR were sent to six participating laboratories. Each laboratory used their own equipment, consumables, and the presumptive reagents conventionally for body fluid (such as acid phosphatase or PSA), DNA extraction, and quantification in practical casework. The reagents and protocol for the 3-plex MSRE-PCR assay and 9 samples were provided by the organizing laboratory. The participating laboratories were requested to fill in the questionnaire after testing. The reported results from all the six participating laboratories were concordant and the expected correct results for the presence of spermatozoa. These outcomes verified the reproducibility and feasibility of the 3-plex MSRE-PCR assay. The results also indicated that the 3-plex MSRE-PCR assay was readily accessible to forensic laboratories for integrating it into current forensic casework processes.

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

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.

Novel identification of biofluids using a multiplex methylation-specific PCR combined with single-base extension system.

PURPOSE: Knowledge of the composition of complex body fluid mixtures may aid forensic investigations greatly. However, many of the traditional tests are presumptive in nature and can lead to ambiguous results. The aim of this study is to establish a reliable method to identify various biofluids via analysis of their DNA methylation profiles. METHODS: A total of eight biofluid-specific methylated markers for saliva, venous blood, vaginal fluids, and semen were isolated from the open database of Infinium HumanMethylation450 BeadChip. These biofluid-specific markers, a control marker to confirm bisulfite conversion, and a gender marker, were combined into a 10-plex methylation-specific PCR single-base-extension (MSP-SBE) system. RESULTS: Analysis of 65 DNA samples isolated from venous blood, semen, vaginal fluid, saliva, and menstrual blood that had been treated with bisulfite, resulted in all eight markers detecting the body fluid to which they were designed. Unambiguous body fluid identification occurred from both single sources of body fluids and complex mixtures. A threshold was devised for each marker to minimize the chance of a false inclusion. The efficacy of the assay and application to forensic practice was demonstrated using five non-probative samples from real alleged sexual assault cases. The system unambiguously determined the biofluid types for the non-probative forensic samples that previously resulted in inconclusive or conflicting results using traditional tests. CONCLUSIONS: The results demonstrated the 10-plex MSP-SBE system established in this study is both sensitive and specific when applied to body fluid identification and can be readily adopted into forensic practice.

Evaluation of three commercial kits effective identification of menstrual blood based on the D-dimer.

Blood or bloodstains are encountered frequently in forensic investigations. Presumptive and more confirmatory tests for peripheral blood are well established, however, similar methods for menstrual blood identification are less so. D-dimer is a fibrin degradation product that occurs at high concentration in menstrual blood and therefore a potential target to screen for this body fluid. We evaluated three rapid tests to determine if they can discriminate menstrual blood from peripheral remote from a laboratory setting. Their sensitivity, specificity and robustness were also assessed. The assays were: a latex agglutination (Dade Dimertest Latex Assay), SERATEC PMB test and OneStep D-dimer RapidCard InstaTest, both of which are based on lateral flow immunochromatographic analysis. Of the three, greater sensitivity was observed using the OneStep D-dimer RapidCard InstaTest, regardless of whether liquid or a stain was used. This test also detected a result using the smallest volume of menstrual blood, 0.003125 µL. Specificity testing was based on six different body fluids (urine, saliva, peripheral blood, semen, sweats and vaginal fluid) resulting in all 30 samples testing negative for the D-dimer using the OneStep D-dimer RapidCard InstaTest. Mixtures at ratios 1:1, 1:3 and 1:9 (menstrual blood: the other biofluid or PBS) were tested and the results showed that D-dimer could be detected for all samples using either the Dade Dimertest Latex Assay or the OneStep D-dimer RapidCard InstaTest. The body fluids were exposed to environmental stresses such as various temperature (-20 °C, 4 °C, room temperature and 37 °C for 30, 90, 180 and 360 days) and fluctuations in humidity (42%, 76% and 100% humidity at room temperature for 1, 3, 5, 10 and 20 days): all samples were D-dimer positive using the OneStep D-dimer RapidCard InstaTest though the strength decreased relative to the increase of storage time and temperature or humidity. All 6 postmortem blood samples gave a positive result for D-dimer using the OneStep D-dimer RapidCard InstaTest and 2 samples gave a positive response using the Dade Dimertest Latex Assay and the SERATEC PMB test; peripheral blood postmortem samples can show an increase in D-dimer. Menstrual blood was recovered from the pads under the sample wells after testing using the two immunochromatographic assays from which STR alleles could be amplified successfully. The results presented here support the application of these commercial kits for effective identification of menstrual blood.

Case report: Coincidental inclusion in a 17-locus Y-STR mixture, wrongful conviction and exoneration.

We report the case of a suspect (Suspect-3) who was convicted (and later exonerated) of participating in the multiple-attacker rape of two women. The forensic evidence against him was his inclusion in a 17-marker Y-STR mixture isolated from semen on one victim's clothing. The DNA inclusion produced a match statistic with a combined probability of inclusion of 1 in 741, and a Likelihood Ratio of 3296. While the defense team was told that Suspect-3 was included in the semen DNA mixture, they were not told that all of the Y-STR alleles could also be explained by just the other two accused attackers' haplotypes. Suspect-3 was subsequently freed after the Taiwan Association for Innocence requested re-examination of the incriminating mixed DNA sample. The Criminal Investigation Bureau was then able to exclude him using an extended set of Y-STR markers (23 loci), leading to his exoneration.

Characterization of the polymorphic repeat sequence within the rDNA IGS of Cannabis sativa.

The rDNA intergenic spacer (IGS) structure of Cannabis sativa contains six variable repeat motifs within a locus spanning 1387 base pairs. The degree of variation of the first three motifs was examined using 77 samples from cannabis samples. The samples originated from five seizures in Taiwan and seed stocks from six different countries. The results showed that there were four types of sequences producing PCR products at either 255, 260, 264 or 265 base pairs. The data obtained indicates that this region of rDNA IGS exhibits a degree of polymorphism that while insufficient by itself can be added to a multiplex with other cannabis STR loci.

Establishing the rDNA IGS structure of Cannabis sativa.

The rDNA intergenic spacer (IGS) structure of Cannabis sativa was established and can be used for classification and identification of this species. In this study, DNA fragments of rDNA IGS were amplified by PCR from Cannabis sativa plant extracts and a 1387 bp fragment was obtained. DNA sequence analysis revealed six different repeat motifs. In the middle of the IGS sequence, there were three sequence motifs, and the same three sections of DNA were then repeated with minor variation in sequence. The terminal region of the IGS was composed of another three different repeat units; multiple copies of these terminal repeat motifs were present in no discernible order. Within six repeat motifs, point variations were observed in five. The DNA sequence of the locus was compared with all the plant sequences registered in GenBank by the Fasta program of GCG software with the result that this DNA fragment was significantly different from any other DNA sequence recorded to date. The most similar sequence was that of Hops (Humulus lupulus), but with a similarity of only 88.9% over 579 bp. These specific and complex variations of IGS may be related to the species and geographic distributions.

Bidens identification using the noncoding regions of chloroplast genome and nuclear ribosomal DNA.

Bidens pilosa L. is a plant producing barbed fruits which, due to its method of seed dispersal, is commonly found during forensic investigations. In Taiwan there are three varieties of the species, radiata, minor and pilosa. Fragments of these three varieties are difficult to differentiate by traditional morphological characteristics and until now little is known of their genetic composition. To discover genetic polymorphisms among these varieties, five loci within the nuclear and chloroplast genomes were screened. A total of 161 specimens were used in this study comprising different geographical populations. Seven samples of Bidens biternata were included as an out-group control. DNA fragments of all samples at the trnL intron and trnL-trnF IGS loci of the chloroplast genome, internal transcribed spacer (ITS1 and ITS2) and the 5.8S of nuclear ribosomal DNA (nrDNA) were amplified and sequenced. There were 3, 4, 20, 12 and 9 sequence types at these five loci, respectively. The sequence types for any locus of trnL intron, ITS1, ITS2 and 5.8S were found to be useful markers to identify Bidens biternata and B. pilosa. The resulting 84 haplotypes at the 5 loci could differentiate the var. radiata from the varieties of B. pilosa with only the exception of 1 type. The genetic polymorphisms can be used when comparing botanical remains to identify the variety of B. pilosa present at a crime scene.