Kelly M. Elkins

Colour quantitation for chemical spot tests for a controlled substances presumptive test database

Crime scene investigators (CSIs) often encounter unknown powders, capsules, tablets, and liquids at crime scenes, many of which are controlled substances. Because most drugs are white powders, however, visual determination of the chemical identity is difficult. Colourimetric tests are a well-established method of presumptive drug identification. Positive tests are often reported differently, however, because two analysts may perceive colour or record colourimetric results in different ways. In addition to perceiving colour differently, it is very common for there to be poor visibility conditions (e.g. rain, darkness) while performing these tests, further obscuring the results. In order to address these concerns and to create uniformity in the reporting of on-site colourimetric test results, this study has evaluated two of the state-of-the-art apps (ColorAssist® and Colorimeter®) for reporting the colour test results quantitatively in red-green-blue (RGB) format. The compiled library database of presumptive test results contains over 3300 data points including over 800 unique drug/test combinations. Variations observed between test replicates, from performing a test on different days, recording with a different device type (e.g. iPod Touch, iPhone models 4, 5c, 5s, or 6), and using different quantities of drug are discussed. Overall, the least variation in Euclidian norm was observed using ColorAssist® with the camera light (25.1±22.1) while the variation between replicates and data recorded using different devices was similar. The resulting library is uploaded to a smartphone application aimed to aid in identifying and interpreting suspected controlled substance evidence. Copyright

Simultaneous Identification of Four "Legal High" Plant Species in a Multiplex PCR High-Resolution Melt Assay().

The international prevalence of “legal high” drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real‐time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high‐resolution melt using LCGreen Plus®. The PCR assay was evaluated based on the following: (i) specificity and selectivity—primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity—serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability—sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging “legal high” species.

The Differentiation of Menstrual from Venous Blood and Other Body Fluids on Various Substrates Using ATR FT-IR Spectroscopy†

Crime scene investigators and laboratory analysts use chemical tests to detect and differentiate body fluids. Testing often requires a sample of the stain, and the chemicals may cause degradation of the fluid or interfere with subsequent tests. Colorimetric chemical tests do not differentiate between different types of the same fluid, such as venous and menstrual blood, and there is no presumptive test available to simultaneously differentiate several body fluids. In this study, we recorded ATR FT‐IR spectra of venous and menstrual blood, semen, saliva, and breastmilk. Neat and simulated casework body fluid samples were analyzed on cotton, nylon, wood, paper, and glass substrates. Differences in fluid composition, including proteins and small molecules, resulted in spectral differences. Venous and menstrual blood is differentiated by the peak at 1039 cm−1 attributed to phosphoric acid found in menstrual blood. Peak intensity is influenced by the porosity and weave of the substrate fabric.

Comparison of proteases in DNA extraction via quantitative polymerase chain reaction.

We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples.

Curriculum and course materials for a forensic DNA biology course

The Forensic Science Education Programs Accreditation Commission (FEPAC) requires accredited programs offer a “coherent curriculum” to ensure each student gains a “thorough grounding of the natural…sciences.” Part of this curriculum includes completion of a minimum of 15 semester‐hours forensic science coursework, nine of which can involve a class in forensic DNA biology. Departments that have obtained or are pursuing FEPAC accreditation can meet this requirement by offering a stand‐alone forensic DNA biology course; however, materials necessary to instruct students are often homegrown and not standardized; in addition, until recently, the community lacked commercially available books, lab manuals, and teaching materials, and many of the best pedagogical resources were scattered across various peer‐reviewed journals. The curriculum discussed below is an attempt to synthesize this disparate information, and although certainly not the only acceptable methodology, the below discussion represents “a way” for synthesizing and aggregating this information into a cohesive, comprehensive whole.

Rapid and inexpensive species differentiation using a multiplex real-time polymerase chain reaction high-resolution melt assay

We demonstrate a method for developing real-time polymerase chain reaction (PCR) high-resolution melt (HRM) assays to identify multiple species present in a mixture simultaneously using LCGreen Plus and melt temperatures. Highly specific PCR primers are designed to yield amplicons with different melt temperatures for simple routine species identification compared with differentiating melt curve kinetics traces or difference plots. This method is robust and automatable, and it leads to savings in time and reagent costs, is easily modified to probe any species of interest, eliminates the need for post-PCR gel or capillary electrophoresis in routine assays, and requires no expensive dye-labeled primers.

Determination of Quality and Quantity of DNA Using Agarose Gel Electrophoresis

This lab will cover agarose gel electrophoresis to visualize a DNA sample of at least ten nanograms using the fluroescent dye SYBR green I. Other dyes including ethidium bromide and PicoGreen may be employed to visualize the DNA. The quality of the recovered DNA is determined by the presence of a high molecular weight, high quality tight band of DNA or a low quality smear of DNA fragments ranging from high to low molecular weight in size. In this experiment, a one percent agarose gel is employed due to its relatively short thirty minute run time. The method is non-destructive and the DNA may be recovered from the gel by excision and purification of the DNA from the gel matrix. The quality and quantity of the recovered DNA will be reported.

Analysis of Deoxyribonucleic Acid (DNA) Sequence Data Using BioEdit

In this experiment, the freeware Biological sequence editor (BioEdit) software or a similar software package will be used to analyze mitochondrial DNA or other sequence electropherograms. Initially, widely-available sequences will be used and may be supplemented by data from the instructor. Base assignments will be verified and any unassigned bases will be called. The readable sequence will be assigned and the presence of a mixture or heteroplasmy will be evaluated. For provided mitochondrial DNA sequences the sequences of the highly variable (HV) I, II, and III regions will be assigned and the differences in the nucleotide sequence will be compared to the revised Cambridge Reference Sequence (rCRS) and noted.

Assessing DNA recovery from chewing gum

The purpose of this study was to evaluate which DNA extraction method yields the highest quantity of DNA from chewing gum. In this study, several popular extraction methods were tested, including Chelex-100, phenol-chloroform-isoamyl alcohol (PCIA), DNA IQ, PrepFiler, and QIAamp Investigator, and the quantity of DNA recovered from chewing gum was determined using real-time polymerase chain reaction with Quantifiler. Chewed gum control samples were submitted by anonymous healthy adult donors, and discarded environmental chewing gum samples simulating forensic evidence were collected from outside public areas (e.g., campus bus stops, streets, and sidewalks). As expected, results indicate that all methods tested yielded sufficient amplifiable human DNA from chewing gum using the wet-swab method. The QIAamp performed best when DNA was extracted from whole pieces of control gum (142.7 ng on average), and the DNA IQ method performed best on the environmental whole gum samples (29.0 ng on average). On average, the QIAamp kit also recovered the most DNA from saliva swabs. The PCIA method demonstrated the highest yield with wet swabs of the environmental gum (26.4 ng of DNA on average). However, this method should be avoided with whole gum samples (no DNA yield) due to the action of the organic reagents in dissolving and softening the gum and inhibiting DNA recovery during the extraction.

Towards the Design of a Cross Platform Solution for Efficient Colorimetric Tests

Every year millions of crime related events are reported, many of which are drug-related. Illegal drugs are among the prime factors for criminal activity, making illicit drug evidence an important type of chemical evidence. Identifying the exact drug is difficult since majority of the seized substances are in the form of white powders. Colorimetric drug tests are used to tentatively identify the substances. We report an automated application using the Android platform for interpretation of presumptive drug test results.