Multiplex high resolution melt (HRM) messenger RNA profiling assays for body fluid identification

Traditional body fluid identification methods use a variety of technologically diverse techniques that do not permit the identification of all body fluids. Definitive identification of the biological material present can be crucial to a fuller understanding of the circumstances pertaining to a crime. Thus definitive molecular based strategies for the conclusive identification of forensically relevant biological fluids need to be developed. Messenger (mRNA) profiling is an example of such a molecular based approach.Current mRNA body fluid identification assays typically involve either capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the time required for analysis. For qRT-PCR assays, only 3 or 4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays and to reduce the time and cost of analysis, we have developed multiplex high resolution melt (HRM) assays that provide an identification of all forensically relevant biological fluids and tissues.     

Application of HRM-PCR (high resolution melting PCR) for identification of forensically important Coleoptera species

Precise estimation time of death is one of key task of forensic entomology. Especially interesting is Coleofauna present at all stages of cadaver decomposition. The morphological identification of Coleoptera species from varying life stages to species level is time-consuming and needs highly qualified entomologists. Among different molecular methods of species identification very promising is high-resolution melting PCR. It allows fast single-tube assignment of analyzed sample to species based on amplicon melting profile. The object of this study were different specimens of Coleoptera collected at pig cadavers in Łomna (central Poland) during 2012 - 2014. Specimes were identified to species by experts of corresponding Coleoptera families. From 120 collected specimens belonging to four families and twelve species HRM-PCR correctly identified specimens belonging to three families and eight species.     

Development of a fast, simple profiling method for sample screening using high resolution melting (HRM) of STRs

A screening assay has been developed to provide preliminary individualization of crime scene samples thus eliminating expensive, time-consuming short tandem repeat (STR) profiling of nonprobative samples. High resolution melting performed in a real-time PCR instrument is used to detect the slight melting differences between the length and sequence variations of 22 forensic STRs. Three STRs (vWA, D18S51, THO1) were chosen to develop an assay which was optimized for Mg++ concentration, annealing/extension time/temperature, assay volume, and bovine serum albumin addition. The assay was tested for reproducibility, uniformity for genotype, melting profile consistency, effects of inhibitors, and mixture effects. The assay could be used to determine DNA concentration when a standard curve is run simultaneously. Calculations of costs show that the assay can save significant time and money for a crime with many samples or suspects.     

Species identification of some common necrophagous flies in Guangdong province, southern China based on the rDNA internal transcribed spacer 2 (ITS2)

Despite the fact that the differences in epidermal ridge density between men and women have been accepted for some time, they have only been thoroughly demonstrated in a small number of populations. The aim of this study is to determine whether such differences exist in a sample of the Spanish population by counting epidermal ridges within three well-defined fingerprint areas. If significant gender differences do exist, then the likelihood of inferring gender from given ridge densities will be explored. The data used in this study was obtained from all 10 fingerprints of 200 individuals of the Spanish Caucasian population (100 males and 100 females) between the ages of 20 and 30. Results show that women tend to have a significantly higher ridge density than men in the distal region of all 10 fingers (radial and ulnar count areas), but not in the proximal region (lower count area). The application of Bayes' theorem, assuming that prior probabilities known for each sex, indicate a threshold for discrimination of sexes.     

Genomic "dactyloscopy" with the use of bacteriophage M13 as a DNA probe (the expertise of material evidence and personal identification)

In this article the authors give a scientific evaluation of genetic dactyloscopy method in which the sites of human chromosomal DNA, possessing structural polymorphism, act as genetic markers. Technology of genome "dactyloscopy" including both the series of standard conventional methods and new methods is presented. The method is highly sensitive and requires small amounts of material for investigation. A practical case is described when genome "dactyloscopy" gave positive results which led to a conclusion on suspect's involvement in the crime.     

An enzyme-linked immunosorbent assay (ELISA) for human semen identification based on a biotinylated monoclonal antibody to a seminal vesicle-specific antigen

Monoclonal antibody mouse antihuman semen-5 (MHS-5) (immunoglobulin G1 [IgG1]) was biotinylated using N-biotinyl-w-aminocaproic acid-N-hydroxysuccinimide ester. This monoclonal antibody-biotin conjugate recognized low molecular weight peptide bands between 10.5 and 20 kilodaltons on immunoblots of liquefied semen. Immunodominant peptides had molecular weights of 10.5, 11.5, and 13.5 kilodaltons. An enzyme-linked immunosorbent assay (ELISA) developed with the biotinylated-MAb and streptavidin peroxidase demonstrated sensitivity curves with lower limits of 10 ng of seminal fluid protein per microtiter well using 50 ng per well of monoclonal antibody-biotin conjugate. Cross-reactivity studies on a panel of human biological fluids and tissues demonstrated no cross-reactivity or false positives using the monoclonal antibody-biotin conjugate. The sensitivity of the monoclonal antibody-biotin ELISA was compared to ELISA based upon a polyclonal secondary antibody-peroxidase conjugate. These findings indicate that this ELISA assay, based on a biotinylated monoclonal antibody to a seminal vesicle-specific antigen, may be useful for semen identification.     

Developmental validation of a novel lateral flow strip test for rapid identification of human blood (Rapid Stain Identification™-Blood)

Human blood is the body fluid most commonly encountered at crime scenes, and blood detection may aid investigators in reconstructing what occurred during a crime. In addition, blood detection can help determine which items of evidence should be processed for DNA-STR testing. Unfortunately, many common substances can cause red-brown stains that resemble blood. Furthermore, many current human blood detection methods are presumptive and prone to false positive results. Here, the developmental validation of a new blood identification test, Rapid Stain Identification™-Blood (RSID™-Blood), is described. RSID™-Blood utilizes two anti-glycophorin A (red blood cell membrane specific protein) monoclonal antibodies in a lateral flow strip test format to detect human blood. We present evidence demonstrating that this test is accurate, reproducible, easy to use, and highly specific for human blood. Importantly, RSID™-Blood does not cross-react with ferret, skunk, or primate blood and exhibits no high-dose hook effect. Also, we describe studies on the sensitivity, body fluid specificity, and species specificity of RSID™-Blood. In addition, we show that the test can detect blood from a variety of forensic exhibits prior to processing for DNA-STR analysis. In conclusion, we suggest that RSID™-Blood is effective and useful for the detection of human blood on forensic exhibits, and offers improved blood detection when compared to other currently used methods.     

Cuticular hydrocarbons as a tool for determining the age of Chrysomya rufifacies (Diptera: Calliphoridae) larvae

Calliphoridae are one of the most important insect groups encountered as evidence collected from a crime scene. Age determination of the immature stages of these necrophagous flies is an important step toward estimating the time of colonization and inferring a minimum postmortem interval (PMI_min) in most instances. To determine if the cuticular hydrocarbons could be used to establish whether the development stages yield characteristics profiles, allowing for age estimation, hydrocarbons were extracted from 1st and 2nd, as well as feeding and post‐feeding 3rd instar Chrysomya rufifacies, the hairy maggot blow fly. Extracted hydrocarbons were analyzed using gas chromatography coupled to mass spectrometry with the aim to investigate the changes in chemical profiles of each larval stage. A total of 23 compounds were identified with most of them being alkanes (65%) with carbon chain lengths of 9–33 carbons, alkenes (18%), and methyl‐branched alkanes (17%). All the hydrocarbons except pentadecane (C15), hexadecane (C16), and nonacosane (C29) showed significant differences in their expression throughout larval development. For 1st instars, nonane was the most abundant (17% of the total hydrocarbons content) compound. Accounting for 11% and 10% of the cuticular hydrocarbons, tricosane and pentacosane, respectively, were the notable hydrocarbons in 2nd instars. For post‐feeding 3rd instars, hentriacontane and tritriacontane were present with relative abundances 18% and 15%, respectively. On average, there was a shift from low to high molecular weight hydrocarbons as the larvae aged. These results indicate the change in hydrocarbons makeup as larvae age and could potentially be used to determine the age of immature C. rufifacies and hence aid in PMI_min estimations. However, future research is needed to validate these results under natural conditions in the field.     

A DNA-based approach for the forensic identification of Asiatic black bear (Ursus thibetanus) in a traditional Asian medicine

Attempts to prevent illegal trade in bile and gallbladders from Asiatic black bears, Ursus thibetanus, are hampered by difficulties associated with identifying such items. We extracted DNA from bile crystals of unknown species origin and generated partial cytochrome b (cyt b) sequences using either universal primers (positioned in conserved regions of cyt b), or primers designed on existing U. thibetanus sequences (UT). Species origin was determined by aligning resolved sequences to reference sequence data. The universal primers were unsuitable for U. thibetanus identification when multiple species templates were present in the samples. The UT primers amplified U. thibetanus DNA from all sample extracts, including those containing mixed species templates. The amplified fragment can distinguish U. thibetanus from the most closely related species, U. americanus, a distinct advantage of DNA sequencing over the methods currently used to analyze suspected U. thibetanus bile.     

Species identification of some common necrophagous flies in Guangdong province,southern China based on the rDNA internal transcribed spacer 2 (ITS2)

Recent studies suggest that sequence analysis technique displays a tempting foreground in identifying unknown specimens of necrophagous flies. In this study, we analyzed 63 complete ITS2 sequences concerning 29 fly species to evaluate the identification potential of the ITS2 region, among of which 41 sequence entries were obtained by sequencing and 22 sequence entries were available on the line. Additionally, phenetic method was recommended to substitute for phylogenetic method because it is very difficult to align the ITS2 sequences. The neighbor-joining tree generated by clustalx1.81 allowed us to differentiate each species. Meanwhile the tree topology also suggested that the ITS2 region showed no resolution for the distinction of geographical populations of some species. The overlapping between intra- and interspecific variation revealed by sequence analyses did not affect species identification. High sequence homology between some congeneric species required further sequencing for forensic practice.     

Phylogenetic relationship of psychoactive fungi based on rRNA gene for a large subunit and their identification using the TaqMan assay (II)

"Magic mushroom (MM)" is the name most commonly given to psychoactive fungi containing the hallucinogenic components: psilocin (1) and psilocybin (2). We investigated the rRNA gene (internal transcribed spacer (ITS) and large subunit (LSU)) of two Panaeolus species and four Psilocybe species fungi (of these, two are non-psilocybin species). On the basis of sequence alignment, we improved the identification system developed in our previous study. In this paper, we describe the new system capable of distinguishing MMs from non-psilocybin Psilocybe species, its application data and the phylogeny of MM species.     

The analysis of biological samples from crime scene for a future human DNA profile confrontation. Effects of presumptive test reagents on the ability to obtain STR profiles for human identification

Because of the increase of evidence of blood stains, that have been washed or cleaned in an attempt to mask the analysis of DNA profiles, there is also an increase in the use of presumptive tests on samples sent to laboratories. Some of the presumptive tests, used to identify blood and semen stains, could potentially affect the recovery of high molecular weight DNA from the samples, or extinguish them, especially those already present in small quantities. After the presumptive tests, often these samples are discarded. This study aimed to examine the possibility of obtaining a DNA profile from samples submitted for presumptive testing and cleaned with bleaches with and without chlorine. Two different protocols were conducted: (a) A unique sample of human blood in natura (5 μL), already typed through the DNA techniques with the genetic profile previously known (control), was distributed onto cotton fabrics and dried at room temperature. Four samples of fabric were macerated in saline solution and Coombs serum and then stored for three months (room temperature and freezer −20 °C). (b) Another sample of human blood, type A, in natura, already typed through the techniques of DNA (control) was used. Aliquots of 200 μL were distributed in: cotton, denim and synthetic fabric. The samples were dried at room temperature for 24 h. The blood stains in those fabrics (cotton, denim and synthetic) were then divided into three groups: unwashed, cleaned with chlorine bleach and cleaned with chlorine bleach and soap powder. The samples were again dried at room temperature for 24 h, before the use of luminol. The DNA were extracted with Chelex 100 and amplified with the Identifiler Kit (Applied Biosystems). The blood stains exposed to saline and Coombs serum had DNA profiles consistent with untreated samples (controls). This result shows that the experts should keep and store the samples treated with saline and Coombs serum for future DNA confrontation when necessary. Also discussed in this paper the pattern of blood stains after washing with bleaching solutions, as well as the quantity of DNA obtained from these samples.     

Preliminary investigations into tris(2,2'-bipyridyl) ruthenium III as a chemiluminescent reagent for the detection of 3,6-diacetylmorphine (heroin) on surfaces

The use of tris(2,2'-bipyridyl) ruthenium (III) as a chemiluminescent spray reagent spot-test for heroin is discussed. Two forms of the reagent are investigated an aqueous and an anhydrous where both were found to give vastly different results. The aqueous reagent giving slow, low intensity chemiluminescence whilst the anhydrous reagent gave a fast, bright response in the presence of 3,6-diacetylmorphine. The anhydrous reagent is less sensitive the slow, intensity response is characteristic of only two opiates tested 3,6-diacetylmorphine and 3-monoacetylmorphine.