Development of Polymerase Chain Reaction-High-Resolution Melt Assay for Waterborne Pathogens Legionella pneumophila, Vibrio parahaemolyticus, and Camplobacter jejuni.

Legionella pneumophila is the waterborne pathogen primarily responsible for causing both Pontiac Fever and Legionnaire's Disease in humans. L. pneumophila is transmitted via aerosolized water droplets. The purpose of this study was to design and test primers to allow for rapid polymerase chain reaction (PCR) melt detection and identification of this infectious agent in cases of clinical or emergency response detection. New PCR primers were designed for this species of bacteria; the primer set was purchased from IDT and the target bacterial DNA was purchased from ATCC. The L. pneumophila primers targeted the macrophage infectivity potentiator gene (mip), which inhibits macrophage phagocytosis. The primers were tested for specificity, repeatability, and sensitivity using PCR-high-resolution melt (HRM) assays. The primer set was found to be specific to the designated bacteria and did not amplify the other twenty-one species from the panel. The L. pneumophila assay was able to be multiplexed. The duplex assay consists of primers for L. pneumophila and Vibrio parahaemolyticus, which are both waterborne pathogens. The triplex assay consists of primers for L. pneumophila, V. parahaemolyticus, and Campylobacter jejuni. The unique melting temperature for the L. pneumophila primer assay is 82.84 ± 0.19 °C, the C. jejuni assay is 78.10 ± 0.58 °C, and the V. parahaemolyticus assay is 86.74 ± 0.65 °C.

Additional predictions for forensic DNA phenotyping of externally visible characteristics using the ForenSeq and Imagen kits.

Multiplex DNA typing methods using massively parallel sequencing can be used to predict externally visible characteristics (EVCs) in forensic DNA phenotyping through the analysis of single-nucleotide polymorphisms. The focus of EVC determination has focused on hair color, eye color, and skin tone as well as visible biogeographical ancestry features. In this study, we researched off-label applications beyond what is currently marketed by the manufacturer of the Verogen ForenSeq kit primer set B and Imagen primer set E SNP loci. We investigated additional EVC predictions by examining published genome wide sequencing studies and reported allele-specific gene expression and predictive values. We have identified 15 SNPs included in the ForenSeq kit panel and Imagen kits that have additional EVC prediction capabilities beyond what is published in the Verogen manuals. The additional EVCs that can be predicted include hair graying, ephelides hyperpigmented spots, dermatoheliosis, facial pigmented spots, standing height, pattern balding, helix-rolling ear morphology, hair shape, hair thickness, facial morphology, eyebrow thickness, sarcoidosis, obesity, vitiligo, and tanning propensity. The loci can be used to augment and refine phenotype predictions with software such as MetaHuman for missing persons, cold case, and historic case investigations.

Pyrosequencing Primer Design for Forensic Biology Applications.

The polymerase chain reaction (PCR) is used to copy DNA in vitro for a variety of applications including amplifying a target DNA, mutating a base, adding tags, and sequencing by synthesis applications. Next-generation sequencing (NGS) is a DNA sequencing technology that has been applied to screening cancer and tissue variants, deep sequencing, and gene expression analysis, and more recently, it has been applied to DNA typing for human identification, estimating age, and detecting and differentiating body fluids. Body fluids are normally identified using color tests, microscopy, and immunochromatographic assays. Pyrosequencing is an NGS approach that has been applied to body fluid analysis. The pyrosequencing assays can detect one or several mixed body fluids by analysis of their tissue-specific differentially methylated regions (tDMRs). Here, the process of designing pyrosequencing primers for forensic biology applications is described.

Comparison of DNA yield and STR profiles from the diaphysis, mid-diaphysis, and metaphysis regions of femur and tibia long bones.

DNA testing of human bones is performed for identification when there is no remaining soft tissue, which often means the samples are old or environmentally compromised. Under these circumstances, it can be difficult to obtain a STR DNA profile. It is important to recover the highest quantity and quality of DNA for STR typing. This study compared the DNA recovery and STR profiles from five anatomical locations in five femora and five tibiae. These locations include the proximal metaphysis, proximal diaphysis, mid-diaphysis, distal diaphysis, and distal metaphysis. Twenty-five femur samples and 25 tibia samples were analyzed using the Qiagen Investigator Quantiplex Pro RGQ Kit for quantitating the extracted DNA and the Qiagen Investigator 24plex QS Kit for STR DNA typing. The highest DNA recovery of the five regions tested in both the femur and the tibia was from the midshaft diaphysis. The femur samples resulted in a significantly higher DNA recovery than the tibia samples as analyzed using a Kruskal-Wallis test (P = 0.002103). The midshaft diaphysis and distal diaphysis yielded the most complete STR DNA profiles in the femora, while the distal and proximal diaphysis yielded the most complete STR DNA profiles in the tibiae. There was no correlation between the amount of DNA recovered and the completeness of the STR DNA profile produced with low template extracts in this study.

Evaluation of Two New Methods for DNA Extraction of "Legal High" Plant Species.

A quick, simple, and high-yield nucleic acid isolation process is crucial for high-quality DNA analysis. The ability of the MicroGEM PDQeX phytoGEM system and Omega Bio-tek E.Z.N.A.® Plant DS Mini kit to extract PCR-ready DNA was evaluated by extracting the forensically relevant "legal high" plant species: Ipomoea purpurea, Artemisia absinthium, Mitragyna speciosa, Datura stramonium, and Papaver somniferum. The plant material was pulverized, processed using the manufacturer's plant protocol for the PDQeX Nucleic Acid Extraction or the manufacturer's protocol for the Omega extraction, quantified using the Invitrogen Qubit 2.0 Fluorometer, and analyzed for amplifiability by PCR using a Qiagen Rotor-Gene Q instrument and published assays. The DNA amplicons for the legal high species produced high-resolution melt curves concordant with the melts observed when DNA was isolated using the Qiagen DNeasy Plant Mini Kit in previous studies.

High Resolution Melt Assays to Detect and Identify Vibrio parahaemolyticus, Bacillus cereus, Escherichia coli, and Clostridioides difficile Bacteria.

Over one hundred bacterial species have been determined to comprise the human microbiota in a healthy individual. Bacteria including Escherichia coli, Bacillus cereus, Clostridioides difficile, and Vibrio parahaemolyticus are found inside of the human body and B. cereus and E. coli are also found on the skin. These bacteria can act as human pathogens upon ingestion of contaminated food or water, if they enter an open wound, or antibiotics, and environment or stress can alter the microbiome. In this study, we present new polymerase chain reaction (PCR) high-resolution melt (HRM) assays to detect and identify the above microorganisms. Amplified DNA from C. difficile, E. coli, B. cereus, and V. parahaemolyticus melted at 80.37 ± 0.45 °C, 82.15 ± 0.37 °C, 84.43 ± 0.50 °C, and 86.74 ± 0.65 °C, respectively. A triplex PCR assay was developed to simultaneously detect and identify E. coli, B. cereus, and V. parahaemolyticus, and cultured microorganisms were successfully amplified, detected, and identified. The assays demonstrated sensitivity, specificity, reproducibility, and robustness in testing.

Detection and Identification of Kratom (Mitragyna speciosa) and Marijuana (Cannabis sativa) by a Real-Time Polymerase Chain Reaction High-Resolution Melt Duplex Assay.

Mitragyna speciosa (MS), a plant commonly known as kratom, is a widely used "legal high" opiate alternative for pain relief. DNA extracted from MS and 26 additional plant species was amplified by PCR using primers targeting the strictosidine beta-D-glucosidase (SGD) and secologanin synthase 2 (SLS2) genes and detected by high-resolution melt curves using three intercalating dyes. Amplicon sizes were confirmed using agarose gel electrophoresis. The observed melt temperatures for SGD and SLS2 were 77.08 ± 0.38°C and 77.61 ± 0.46°C, respectively, using SYBR® Green I; 80.18 ± 0.27°C and 80.59 ± 0.08°C, respectively, using Radiant™ Green; and 82.19 ± 0.04°C and 82.62 ± 0.13°C, respectively, using the LCGreen® PLUS dye. The SLS2 primers demonstrated higher specificity and identified MS DNA at 0.05 ng/µL. In a duplex reaction, SLS2 and tetrahydrocannabinoic acid synthase gene primers detected and differentiated MS and Cannabis sativa (CS) by melt peaks at 82.63 ± 0.35°C and 85.58 ± 0.23°C, respectively, using LCGreen® PLUS.

Development of a PCR High-Resolution Melt Assay for Artemisia absinthium (Wormwood) and a Triplex Assay with Two Additional "Unregulated Legal High" Species Datura stramonium (Jimson Weed) and Merremia tuberosa (Hawaiian Woodrose).

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an "unregulated legal high" in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be "thujone-free." However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) high-resolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three "unregulated legal high" species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42 ± 0.20°C, 83.88 ± 0.22°C, and 87.77 ± 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed.

Detection and Identification of Psilocybe cubensis DNA Using a Real-Time Polymerase Chain Reaction High Resolution Melt Assay.

Psilocybe cubensis, or "magic mushroom," is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web-based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy® Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR® Green I, Radiant™ Green, or LCGreen Plus® intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR® Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant™ Green dye.

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.

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.

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.

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 © 2016 John Wiley & Sons, Ltd.

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.

Primer design for PCR reactions in forensic biology.

The polymerase chain reaction (PCR) is a popular method to copy DNA in vitro. Its invention revolutionized fields ranging from clinical medicine to anthropology, molecular biology, and forensic biology. The method employs one of many available heat-stable DNA polymerases in a reaction that is repeated many times in situ. The DNA polymerase reads a template DNA strand and using the components of the reaction mix, catalyzes the addition of free 2'-deoxynucleotide triphosphate nitrogenous bases to short segment of DNA that forms a complement with the template via Watson-Crick base pairing. This short segment of DNA is referred to as a PCR primer and it is essential to the success of the reaction. The most widely used application of PCR in forensic labs is the amplification of short tandem repeat (STR) loci used in DNA typing. The STRs are routinely evaluated in concert with 16 or more reactions, a multiplex, run in one test tube simultaneously. In a multiplex, it is essential that the primers work specifically and accurately on the intended reactions without hindering the other reactions. The primers, which are very specific, also can be used to probe single nucleotide polymorphisms (SNPs) in a DNA sequence of interest by single base extension. Primers are often designed using one of many available automated software packages. Here the process of manually designing PCR primers for forensic biology using no-cost software is described.

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.

Fluorescence characterization of the interaction Suwannee river fulvic acid with the herbicide dichlorprop (2-(2,4-dichlorophenoxy)propionic acid) in the absence and presence of aluminum or erbium.

This study uses fluorescence spectroscopy to better understand the role of environmental metal ions in the interaction of charged herbicides with biochemical degradation product Suwannee River fulvic acid (SRFA). The interactions between the widely-used herbicide dichlorprop (2-(2,4-dichlorophenoxy)propionic acid) (DCPPA) with Al(3+) and the comparative metal Er(3+) were probed at pH 4.0. Fluorescence experiments on binary solutions at pH 4.0 clearly indicated that Al(3+) and Er(3+) strongly interact with both SRFA and DCPPA alone in solution as demonstrated by fluorescence quenching with DCPPA and enhancement with SRFA by Al(3+) and fluorescence quenching of both SRFA and DCPPA fluorescence by Er(3+). Titrating Al(3+) or Er(3+) to SRFA-DCPPA quenched SRFA fluorescence as compared to the SRFA-metal ion binary complexes. Formation constants were determined using the Ryan-Weber model for the titration data. The DCPPA fluorescence results strongly support the formation of DCPPA-Al(3+) and DCPPA-Er(3+) complexes at pH values above the pK(a) (3.0) of DCPPA. Excitation and emission data obtained on ternary solutions of SRFA-Al(3+)-DCPPA and SRFA-Er(3+)-DCPPA complexes at pH 4.0 suggest that at this pH where the predominant DCPPA species is negatively-charged, Al(3+) and Er(3+) metal ions may function to "bridge" negatively-charged fulvic acids to negatively-charged pesticides. Fluorescence data collected on UV-irradiated ternary complexes indicate that both metals can also bridge DCPPA interactions with SRFA under those conditions. The results of our studies suggest that creation of a herbicide-free boundary corridor is recommended near mines and runoff areas with metal ions in surface waters to control possible complexation among fulvic acids, DCPPA and metal ions that maintains these molecules in a bioavailable state to plants and animals.