ABSTRACT
Ricin, a toxic protein from the castor plant, is of forensic and biosecurity interest because of its high toxicity and common occurrence in crimes and attempted crimes. Qualitative methods to detect ricin are therefore needed. Untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics methods are well suited because of their high specificity. Specificity in LC-MS/MS comes from both the LC and MS components. However, modern untargeted proteomics methods often use nanoflow LC, which has less reproducible retention times than standard-flow LC, making it challenging to use retention time as a point of identification in a forensic assay. We address this challenge by using retention times relative to a standard, namely, the uniformly 15N-labeled ricin A-chain produced recombinantly in a bacterial expression system. This material, added as an internal standard prior to trypsin digestion, produces a stable-isotope-labeled standard for every ricin tryptic peptide in the sample. We show that the MS signals for 15N and natural isotopic abundance ricin peptides are distinct, with mass shifts that correspond to the numbers of nitrogen atoms in each peptide or fragment. We also show that, as expected, labeled and unlabeled peptides coelute, with relative retention time differences of less than 0.2%.
Subject(s)
Chromatography, Liquid/methods , Forensic Sciences/methods , Isotope Labeling , Nanotechnology/methods , Ricin/chemistry , Tandem Mass Spectrometry/methods , Nitrogen Isotopes , Recombinant ProteinsABSTRACT
Ricin, a protein found in castor seeds, is a lethal toxin that is designated as a category 2 select agent, and cases of attempted ricin poisoning are relatively common. Many methods to detect protein toxins such as ricin use targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify toxin peptides, usually tryptic peptides. The successful use of untargeted methods has also been reported. However, the use of untargeted proteomics methods, including database search, for peptide and protein identification is less common in forensic practice and may be unfamiliar to forensic science practitioners. Here, we propose a method to create spectral libraries of tryptic ricin peptides and use these libraries for ricin identification by spectral library search, which may be more familiar to forensic scientists because of the use of spectral libraries in small molecule identification. Peptide spectral libraries offer a direct comparison to an authentic standard, a key element of forensic analysis, but have not previously been used in a forensic context. To construct these spectral libraries, two pure ricin samples (one from a proposed standard reference material) were digested with trypsin and analyzed using a standard shotgun LC-MS/MS protocol. Spectral libraries were created from resulting tryptic peptides identified from filtered search results from four database search tools. The library was then used in a search using SpectraST on forensically realistic castor seed extracts. These castor seed samples were made using the crude methods commonly encountered in real-world ricin cases. Analysis showed that the spectral library search resulted in more peptides identified from crude castor seed samples compared to MS-GF+ and Sequest plus Percolator database searches. These results, the first published use of spectral library search to detect protein toxins in forensically relevant samples, suggest that computational comparison of putative ricin peptide spectra to library spectra can be an effective method to detect ricin in an unknown sample. Data are available via ProteomeXchange with identifier PXD013711.
Subject(s)
Chromatography, Liquid/methods , Peptide Library , Peptides/metabolism , Proteomics/methods , Ricin/metabolism , Tandem Mass Spectrometry/methods , Computational Biology/methods , Forensic Medicine/methods , Humans , Reproducibility of Results , Ricin/isolation & purification , Software , Trypsin/metabolismABSTRACT
Robust and highly specific methods for the detection of the protein toxin ricin are of interest to the law enforcement community. In previous studies, methods based on liquid chromatography-tandem mass spectrometry shotgun proteomics have been proposed. The successful implementation of this approach relies on specific data evaluation criteria addressing (1) the quality of the mass spectrometric data, (2) the confidence of peptide identifications (peptide-spectrum matches), and (3) the number and sequence specificity of peptides detected. We present such data evaluation criteria and use a novel approach to establish the limit of detection for this ricin assay. Specifically, we use logistic regression to determine the probability of detection for individual ricin peptides at different concentrations. We then apply basic rules from probability theory, combining these individual peptide probabilities into an overall assay limit of detection. This procedure yields an assay limit of detection for ricin at 42.5 ng on column or 21.25 ng/µL for a 2-µL injection. We also show that, despite the conventional wisdom that detergents are deleterious to mass spectrometric analyses, the presence of Tween-20 did not prevent detection of ricin peptides, and indeed assays performed in buffers that included Tween-20 gave better results than assays performed using other buffer formulations with or without detergent removal.
Subject(s)
Limit of Detection , Proteomics/methods , Ricin/analysis , Amino Acid Sequence , Polysorbates/chemistry , Ricin/chemistry , Ricin/metabolismABSTRACT
Mass spectrometry-based proteomics has been a useful tool for addressing numerous questions in basic biology research for many years. This success, combined with the maturity of mass spectrometric instrumentation, the ever-increasing availability of protein sequence databases derived from genome sequencing, and the growing sophistication of data analysis methods, places proteomics in a position to have an important role in biological forensics. Because proteins contain information about genotype (sequence) and phenotype (expression levels), proteomics methods can both identify biological samples and characterize the conditions that produced them. In addition to serving as a valuable orthogonal method to genomic analyses, proteomics can be used in cases where nucleic acids are absent, degraded, or uninformative. Mass spectrometry provides both broad applicability and exquisite specificity, often without customized detection reagents like primers or antibodies. This review briefly introduces proteomics methods, and surveys a variety of forensic applications (including criminal justice, historical, archaeological, and national security areas). Finally, challenges and crucial areas for further research are addressed.
Subject(s)
Forensic Sciences , Proteomics , Archaeology , Body Fluids/metabolism , Bone and Bones/metabolism , Chromatography , Doping in Sports , Food , Hair/metabolism , Humans , Mass Spectrometry , Microbiota , Peptides/analysis , Proteolysis , Proteome , Sequence Analysis, Protein , Species Specificity , Toxins, Biological/metabolismABSTRACT
Microorganisms alter gene and protein expression in response to environmental conditions to adapt and survive. Whereas the genetic composition of a microbe represents an organism's biological potential, the proteins expressed provide a functional readout of the organism's response to the environment. Understanding protein expression patterns in response to specific environmental conditions furthers fundamental knowledge about a microbe, which can be especially useful for understudied organisms such as Clostridium botulinum examined herein. In addition, protein expression patterns that reproducibly occur in certain growth conditions hold potential in fields such as microbial forensics, in which determination of conditions in which an unknown possible biothreat sample had been grown may be important. To investigate the identity and reproducibility of protein profile patterns for varied strains, we defined the proteomic profiles of four Group I strains of Clostridium botulinum, a Category A biothreat agent and the organism responsible for the production of the botulinum neurotoxin (BoNT), in two different culture media grown for five days. The four C. botulinum strains produced one of three neurotoxins (BoNT/A, /B, or /F), and their protein profiles were compared to that of a fifth non-toxigenic strain of C. sporogenes. These strains each had DNA sequences available to assist in accurate protein identification. Differing culture growth phase, bacterial strain, and growth medium resulted in reproducible protein profiles, which were used to calculate relative protein abundance ratios as an internally normalized metric of microbial growth in varying conditions. The resulting protein profiles provide functional information about how four Group I C. botulinum strains and a C. sporogenes strain respond to the culture environment during growth and explores the feasibility of using these proteins to characterize unknown samples.
Subject(s)
Botulinum Toxins/metabolism , Clostridium botulinum/metabolism , Botulinum Toxins/genetics , Cell Culture Techniques , Clostridium botulinum/genetics , Clostridium botulinum/growth & development , Culture Media/analysis , Gene Expression , Phylogeny , Polymorphism, Single Nucleotide , Proteome , Proteomics , Species SpecificityABSTRACT
The toxic protein ricin (also known as RCA60), found in the seed of the castor plant (Ricinus communis) is frequently encountered in law enforcement investigations. The ability to detect ricin by analyzing its proteolytic (tryptic) peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is well established. However, ricin is just one member of a family of proteins in R. communis with closely related amino acid sequences, including R. communis agglutinin I (RCA120) and other ricin-like proteins (RLPs). Inferring the presence of ricin from its constituent peptides requires an understanding of the specificity, or uniqueness to ricin, of each peptide. Here we describe the set of ricin-derived tryptic peptides that can serve to uniquely identify ricin in distinction to closely-related RLPs and to proteins from other species. Other ricin-derived peptide sequences occur only in the castor plant, and still others are shared with unrelated species. We also characterized the occurrence and relative abundance of ricin and related proteins in an assortment of forensically relevant crude castor seed preparations. We find that whereas ricin and RCA120 are abundant in castor seed extracts, other RLPs are not represented by abundant unique peptides. Therefore, the detection of peptides shared between ricin and RLPs (other than RCA120) in crude castor seed extracts most likely reflects the presence of ricin in the sample.
Subject(s)
Chemical Warfare Agents/analysis , Ricin/analysis , Ricinus communis/chemistry , Amino Acid Sequence , Chemical Warfare Agents/chemistry , Chromatography, Liquid , Peptides/analysis , Plant Extracts/chemistry , Plant Proteins/analysis , Ricin/chemistry , Seeds/chemistry , Tandem Mass SpectrometryABSTRACT
Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Analysis of cellular proteins is dependent upon efficient extraction from bacterial samples, which can be challenging with increasing complexity and refractory characteristics. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrichment for certain classes of proteins. The method presented here is technically simple, does not require specialized equipment such as a mechanical disrupter, and is effective for protein extraction of the particularly challenging sample type of Bacillus anthracis Sterne spores. The ability of Trichloroacetic acid (TCA) extraction to isolate proteins from spores and enrich for spore-specific proteins was compared to the traditional mechanical disruption method of bead beating. TCA extraction improved the total average number of proteins identified within a sample as compared to bead beating (547 vs 495, respectively). Further, TCA extraction enriched for 270 spore proteins, including those typically identified by first isolating the spore coat and exosporium layers. Bead beating enriched for 156 spore proteins more typically identified from whole spore proteome analyses. The total average number of proteins identified was equal using TCA or bead beating for easily lysed samples, such as B. anthracis vegetative cells. As with all assays, supplemental methods such as implementation of an alternative preparation method may simplify sample preparation and provide additional insight to the protein biology of the organism being studied.
Subject(s)
Bacillus anthracis/chemistry , Bacterial Proteins/analysis , Bacterial Proteins/isolation & purification , Proteome/analysis , Proteome/isolation & purification , Proteomics/methods , Spores, Bacterial/chemistry , Bacillus anthracis/drug effects , Spores, Bacterial/drug effects , Trichloroacetic Acid/metabolismABSTRACT
We analyzed 21 neat acetone samples from 15 different suppliers to demonstrate the utility of a coupled stable isotope and trace contaminant strategy for distinguishing forensically-relevant samples. By combining these two pieces of orthogonal data we could discriminate all of the acetones that were produced by the 15 different suppliers. Using stable isotope ratios alone, we were able to distinguish 8 acetone samples, while the remaining 13 fell into four clusters with highly similar signatures. Adding trace chemical contaminant information enhanced discrimination to 13 individual acetones with three residual clusters. The acetones within each cluster shared a common manufacturer and might, therefore, not be expected to be resolved. The data presented here demonstrates the power of combining orthogonal data sets to enhance sample fingerprinting and highlights the role disparate data could play in future forensic investigations.
Subject(s)
Acetone/isolation & purification , Forensic Sciences/methods , Mass Spectrometry/statistics & numerical data , Acetone/classification , Carbon Isotopes , Chemical Terrorism/prevention & control , Deuterium , Discriminant Analysis , Forensic Sciences/instrumentation , Hexanones/isolation & purification , Humans , Ketones/isolation & purification , Pentanols/isolation & purification , Pentanones/isolation & purificationABSTRACT
Here we demonstrate that when Yersinia pesitis is grown in laboratory media, peptides from the medium remain associated with cellular biomass even after washing and inactivation of the bacteria by different methods. These peptides are characteristic of the type of growth medium and of the manufacturer of the medium, reflecting the specific composition of the medium. We analyzed biomass-associated peptides from cultures of two attenuated strains of Yersinia pestis [KIM D27 (pgm-) and KIM D1 (lcr-)] grown in several formulations of 4 different media (tryptic soy broth (TSB), brain-heart infusion (BHI), Luria-Bertani broth (LB), and glucose (G) medium) made from components purchased from different suppliers. Despite the range of growth medium sources and the associated manufacturing processes used in their production, a high degree of peptide similarity was observed for a given medium recipe; however, notable differences in the termination points of select peptides were observed in media formulated using products from some suppliers, presumably reflecting the process by which a manufacturer performed protein hydrolysis for use in culture media. These results may help explain the presence of peptides not explicitly associated with target organisms during proteomic analysis of microbes and other biological systems that require culturing. While the primary aim of this work is to outline the range and type of medium peptides associated with Yersinia pestis biomass and improve the quality of proteomic measurements, these peptides may also represent a potentially useful forensic signature that could provide information about microbial culturing conditions.
Subject(s)
Bacterial Proteins/isolation & purification , Culture Media/chemistry , Peptides/isolation & purification , Proteomics/standards , Yersinia pestis/metabolism , Adsorption , Amino Acid Sequence , Bacterial Proteins/metabolism , Chromatography, Liquid , Mass Spectrometry , Molecular Sequence Data , Peptides/metabolism , Yersinia pestis/growth & developmentABSTRACT
The investigation of crimes involving chemical or biological agents is infrequent, but presents unique analytical challenges. The protein toxin ricin is encountered more frequently than other agents and is found in the seeds of Ricinus communis, commonly known as the castor plant. Typically, the toxin is extracted from castor seeds utilizing a variety of different recipes that result in varying purity of the toxin. Moreover, these various purification steps can also leave or differentially remove a variety of exogenous and endogenous residual components with the toxin that may indicate the type and number of purification steps involved. We have applied three gas chromatography-mass spectrometry (GC-MS) based analytical methods to measure the variation in seed carbohydrates and castor oil ricinoleic acid, as well as the presence of solvents used for purification. These methods were applied to the same samples prepared using four previously identified toxin preparation methods, starting from four varieties of castor seeds. The individual data sets for seed carbohydrate profiles, ricinoleic acid, or acetone amount each provided information capable of differentiating different types of toxin preparations across seed types. However, the integration of the data sets using multivariate factor analysis provided a clear distinction of all samples based on the preparation method, independent of the seed source. In particular, the abundance of mannose, arabinose, fucose, ricinoleic acid, and acetone were shown to be important differentiating factors. These complementary tools provide a more confident determination of the method of toxin preparation than would be possible using a single analytical method.
Subject(s)
Analytic Sample Preparation Methods/methods , Gas Chromatography-Mass Spectrometry/methods , Ricin/analysis , Systems Integration , Acetone/analysis , Acetone/chemistry , Hydrogen-Ion Concentration , Monosaccharides/analysis , Monosaccharides/chemistry , Multivariate Analysis , Ricin/chemistry , Ricin/isolation & purification , Ricinoleic Acids/analysis , Ricinoleic Acids/chemistry , Ricinus/chemistry , Ricinus/enzymology , Seeds/chemistry , Seeds/enzymologyABSTRACT
Biological materials generally require stabilization to retain activity or viability in a dry form. A number of industrial products, such as vaccines, probiotics and biopesticides have been produced as dry preparations. The same methods and materials used for stabilizing commercial microbial products may be applicable to preserving biothreat pathogens in a dry form. This is a likely step that may be encountered when looking at samples from terrorism attempts since only spores, such as those from Bacillus anthracis, are inherently stable when dried. The stabilizers for microbial preparations generally include one or more small carbohydrates. Different formulations have been reported for different industrial products and are often determined empirically. However sugar alcohols (mannitol and sorbitol) and disaccharides (lactose, sucrose and trehalose) are the common constituents of these formulations. We have developed an analytical method for sample preparation and detection of these simple carbohydrates using two complementary analytical tools, MALDI-MS and GC-MS. The native carbohydrates and other constituents of the formulation are detected by MALDI-MS as a screening tool. A longer and more detailed analysis is then used to specifically identify the carbohydrates by derivatization and GC-MS detection. Both techniques were tested against ten different types of stabilization recipes with Yersinia pestis cell mass cultured on different media types used as the biological component. A number of additional components were included in these formulations including proteins and peptides from serum or milk, polymers (e.g. poly vinyl pyrrolidone - PVP) and detergents (e.g. Tween). The combined method was characterized to determine several figures of merit. The accuracy of the method was 98% for MALDI-MS and 100% for GC-MS. The repeatability for detection of carbohydrates by MALDI-MS was determined to be 96%. The repeatability of compound identification by GC-MS was determined by monitoring variation in retention time, which is vital for identification of isomeric carbohydrates. The figures of merit illustrate an effective and accurate method for mono and disaccharide detection independent of formulation. This meets our primary goal for method development as small carbohydrates are among the most common stabilizers employed.
ABSTRACT
As a preservation solution, (1%) ammonium chloride may be preferred over other conventionally used storage solutions because of its compatibility with analytical techniques such as Mass Spectrometry. In this study, ammonium chloride performed as well or better than phosphate buffered saline with Tween or Butterfields/Tween for preserving Francisella tularensis subsp. novicida.
Subject(s)
Forensic Medicine/methods , Francisella tularensis/isolation & purification , Microbial Viability , Preservation, Biological/methods , Specimen Handling/methods , Ammonium Chloride/metabolism , HumansABSTRACT
Samples containing the toxic castor bean protein ricin have been recently seized in connection with biocriminal activity. Analytical methods that enable investigators to determine how the samples were prepared and to match seized samples to potential source materials are needed. One commonly described crude ricin preparation method is acetone extraction of crushed castor beans. Here, we describe the use of solid-phase microextraction and headspace analysis to determine whether castor beans were processed by acetone extraction. We prepared acetone-extracted castor bean mash, along with controls of unextracted mash and mash extracted with nonacetone organic solvents. Samples of acetone-extracted mash and unextracted mash were stored in closed containers for up to 109 days at both room temperature and -20 degrees C, and in open containers at room temperature for up to 94 days. Acetone-extracted bean mash could consistently be statistically distinguished from controls, even after storage in open containers for 94 days.
Subject(s)
Acetone/isolation & purification , Ricin , Solvents/isolation & purification , Ricinus communis , Gas Chromatography-Mass Spectrometry , Hot Temperature , Solid Phase Microextraction , Specimen Handling , TemperatureABSTRACT
Presented here is an analytical method to detect residual agar from a bacterial spore sample as an indication of culturing on an agar plate. This method is based on the resolubilization of agar polysaccharide from a bacterial spore sample, enzymatic digestion, followed by electrospray ionization tandem mass spectrometry (ESI-MS(n)) analysis for detection of a specific agar fragment ion. A range of Bacillus species and strains were selected to demonstrate the effectiveness of this approach. The characteristic agar fragment ion was detected in the spores grown on agar that were washed from 1 to 5 times, irradiated or nonirradiated, and not in the spores grown in broth. A sample containing approximately 10(8) spores is currently needed for confident detection of residual agar from culture on agar plates in the presence of bacterial spores with a limit of detection of approximately 1 ppm agar spiked into a broth-grown spore sample. The results of a proficiency test with 42 blinded samples are presented demonstrating the utility of this method with no false positives and only three false negatives for samples that were below the detection level of the method as documented.
Subject(s)
Agar/analysis , Spores, Bacterial/chemistry , Agar/metabolism , Bacillus/growth & development , Bacillus/metabolism , Culture Media/chemistry , Culture Techniques , Spectrometry, Mass, Electrospray Ionization , Spores, Bacterial/metabolismABSTRACT
This paper highlights the distinctions between the infrared (IR) absorption spectra of vegetative versus sporulated Bacillus bacteria. It is observed that there are unique signatures clearly associated with either the sporulated or vegetative state and that vegetative cells (or cell debris) can contribute to the spore spectra. A distinct feature at approximately 1739 cm(-1) appears to be unique to vegetative cell spectra and can also be used as an indicator of vegetative cells or cell debris in the spore spectra. The data indicate that the band arises from a lipid-soluble species such as an ester or phospholipid carbonyl bond and are consistent with it being either phosphatidyl glycerol (PG) or phosphatidylethanolamine (PE), two major classes of phospholipids found in vegetative cells of Bacillus species. A companion work discusses bands associated with the sporulated state.
Subject(s)
Bacillus/chemistry , Phospholipids/chemistry , Spectrophotometry, Infrared/methods , Bacillus/cytology , Phosphatidylethanolamines/chemistry , Phosphatidylglycerols/chemistry , Spores, Bacterial/chemistryABSTRACT
Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for characterization and analysis of microorganisms, specifically bacteria, is described here as a rapid screening tool. The objective of this technique is not comprehensive protein analysis of a microorganism but rather a rapid screening of the organism and the accessible protein pattern for characterization and distinction. This method is based on the ionization of the readily accessible and easily ionizable portion of the protein profile of an organism that is often characteristic of different bacterial species. The utility of this screening approach is yet to reach its full potential but could be applied to food safety, disease outbreak monitoring in hospitals, culture stock integrity and verification, microbial forensics, or homeland security applications.
Subject(s)
Bacteria/chemistry , Bacteria/isolation & purification , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Animals , Bacillus subtilis/chemistry , Bacillus subtilis/cytology , Bacillus subtilis/isolation & purification , Bacteria/classification , Bacteria/cytology , Spores, Bacterial/chemistryABSTRACT
Detection of small quantities of agar associated with spores of Bacillus anthracis could provide key information regarding its source or growth characteristics. Agar, widely used in growth of bacteria on solid surfaces, consists primarily of repeating polysaccharide units of 3,6-anhydro-l-galactose (AGal) and galactose (Gal) with sulfated and O-methylated galactoses present as minor constituents. Two variants of the alditol acetate procedure were evaluated for detection of potential agar markers associated with spores. The first method employed a reductive hydrolysis step, to stabilize labile anhydrogalactose, by converting to anhydrogalactitol. The second eliminated the reductive hydrolysis step simplifying the procedure. Anhydrogalactitol, derived from agar, was detected using both derivatization methods followed by gas chromatography-mass spectrometry (GC-MS) analysis. However, challenges with artifactual background (reductive hydrolysis) or marker destruction (hydrolysis) respectively lead to the use of an alternative agar marker. A minor agar component, 6-O-methyl galactose (6-O-M gal), was readily detected in agar-grown but not broth-grown bacteria. Detection was optimized by the use of gas chromatography-tandem mass spectrometry (GC-MS-MS). With appropriate choice of sugar marker and analytical procedure, detection of sugar markers for agar has considerable potential in microbial forensics.
Subject(s)
Agar/analysis , Bacillus anthracis/growth & development , Culture Media/chemistry , Spores, Bacterial/chemistry , Carbohydrates/analysis , Gas Chromatography-Mass Spectrometry , Methylgalactosides/analysisABSTRACT
In the aftermath of the 2001 anthrax letters, researchers have been exploring ways to predict the production environment of unknown-source microorganisms. Culture medium, presence of agar, culturing temperature, and drying method are just some of the broad spectrum of characteristics an investigator might like to infer. The effects of many of these factors on microorganisms are not well understood, but the complex way in which microbes interact with their environments suggests that numerous analytical techniques measuring different properties will eventually be needed for complete characterization. In this work, we present a Bayesian statistical framework for integrating disparate analytical measurements. We illustrate its application to the problem of characterizing the culture medium of Bacillus spores using three different mass spectral techniques. The results of our study suggest that integrating data in this way significantly improves the accuracy and robustness of the analyses.
Subject(s)
Bacillus anthracis/chemistry , Bacillus thuringiensis/chemistry , Chemistry Techniques, Analytical/methods , Mass Spectrometry , Spores, Bacterial/chemistry , Bayes Theorem , Culture Media/chemistryABSTRACT
We demonstrate the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) in a forensics application to distinguish Bacillus subtilis spores grown in various media based on the elemental signatures of the spores. Triplicate cultures grown in each of four different media were analyzed to obtain TOF-SIMS signatures comprised of 16 elemental intensities. Analysis of variance was unable to distinguish growth medium types based on 40Ca-normalized signatures of any single normalized element. Principal component analysis proved successful in separating the spores into groups consistent with the media in which they were prepared. Confusion matrices constructed using nearest-neighbor classification of the PCA scores confirmed the predictive utility of TOF-SIMS elemental signatures in identifying sporulation medium. Theoretical calculations based on the number and density of spores in an analysis area indicate an analytical sample size of about 1 ng, making this technique an attractive method for bioforensics applications.