Three-Dimensional-Printed Instrument for Isothermal Nucleic Acid Amplification with Real-Time Colorimetric Imaging.

Isothermal amplification methods have become popular in research due to the simplicity of the technology needed to run the reactions. Specifically, loop-mediated isothermal amplification (LAMP) has been widely used for various applications since first reported in 2000. LAMP reactions are commonly monitored with the use of colorimetry. Although color changes associated with positive amplification are apparent to the naked eye, this detection method is subjective due to inherent differences in visual perception from person to person. The objectivity of the colorimetric detection method may be improved by programmed image capture over time with simultaneous heating. As such, the development of a novel, one-step, automated, and integrated analysis system capable of performing these tasks in parallel is detailed herein. The device is adaptable to multiple colorimetric dyes, cost-effective, 3D-printed for single-temperature convective heating, and features an easy-to-use LabVIEW software program developed for automated image analysis. The device was optimized and subsequently validated using four messenger-RNA targets and mock forensic samples. The performance of our device was determined to be comparable to that of a conventional thermal cycler and smartphone image analysis, respectively. Moreover, the outlined system is capable of objective colorimetric analysis, with exceptional throughput of up to 96 samples at once.

Rapid Microchip Electrophoretic Separation of Novel Transcriptomic Body Fluid Markers for Forensic Fluid Profiling.

Initial screening of criminal evidence often involves serological testing of stains of unknown composition and/or origin discovered at a crime scene to determine the tissue of origin. This testing is presumptive but critical for contextualizing the scene. Here, we describe a microfluidic approach for body fluid profiling via fluorescent electrophoretic separation of a published mRNA panel that provides unparalleled specificity and sensitivity. This centrifugal microfluidic approach expedites and automates the electrophoresis process by allowing for simple, rotationally driven flow and polymer loading through a 5 cm separation channel; with each disc containing three identical domains, multi-sample analysis is possible with a single disc and multi-sample detection per disc. The centrifugal platform enables a series of sequential unit operations (metering, mixing, aliquoting, heating, storage) to execute automated electrophoretic separation. Results show on-disc fluorescent detection and sizing of amplicons to perform comparably with a commercial 'gold standard' benchtop instrument and permitted sensitive, empirical discrimination between five distinct body fluids in less than 10 min. Notably, our microfluidic platform represents a faster, simpler method for separation of a transcriptomic panel to be used for forensically relevant body fluid identification.

An ultrafast SARS-CoV-2 virus enrichment and extraction method compatible with multiple modalities for RNA detection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic RNA virus characterized by high transmission rates and pathogenicity worldwide. Continued control of the COVID-19 pandemic requires the diversification of rapid, easy to use, sensitive, and portable methods for SARS-CoV-2 sample preparation and analysis. Here, we propose a method for SARS-CoV-2 viral enrichment and enzymatic extraction of RNA from clinically relevant matrices in under 10 min. This technique utilizes affinity-capture hydrogel particles to concentrate SARS-CoV-2 from solution, and leverages existing PDQeX technology for RNA isolation. Characterization of our method is accomplished with reverse transcription real-time polymerase chain reaction (RT-PCR) for relative, comparative RNA detection. In a double-blind study analyzing viral transport media (VTM) obtained from clinical nasopharyngeal swabs, our sample preparation method demonstrated both comparable results to a routinely used commercial extraction kit and 100% concordance with laboratory diagnoses. Compatibility of eluates with alternative forms of analysis was confirmed using microfluidic RT-PCR (µRT-PCR), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP). The alternative methods explored here conveyed successful amplification from all RNA eluates originating from positive clinical samples. Finally, this method demonstrated high performance within a saliva matrix across a broad range of viral titers and dilutions up to 90% saliva matrix, and sets the stage for miniaturization to the microscale.

Optimization of novel loop-mediated isothermal amplification with colorimetric image analysis for forensic body fluid identification.

Accurate presumptive and confirmatory test use for forensic body fluid identification is essential for gaining contextual information for crime scene investigators. Loop-mediated isothermal amplification (LAMP) is an ideal method for forensic body fluid identification because it is highly specific and generates multi-sized amplicon DNA, and successful amplification results can be read out colorimetrically. Here, we show preliminary data on a LAMP method that rapidly identifies body fluids including venous blood, semen, and saliva, based on colorimetric response and image analysis. The method is designed for easy implementation into forensic casework protocols with minimal disruption to DNA analysis. LAMP naturally increases target specificity due to the use of multiple primers for one target and mRNA targets were used for tissue and human specificity. With colorimetric detection as an inherent part of LAMP, samples that are positive or negative for any of the body fluids are readily identified by image capture and analysis, thus eliminating subjectivity. Results show by using the 3D-printed imaging system specific color ranges can be set for easy determination of body fluids. The resulting color change can be seen in <30 min using a universal temperature and primer concentration for all body fluids. This simple method and imaging system allow for minimal hands-on time with objective image analysis and presents a pathway for creating a new potential method for forensic body fluid identification.

Comparative Evaluation and Quantitative Analysis of Loop-Mediated Isothermal Amplification Indicators.

Loop-mediated isothermal amplification (LAMP) as a diagnostic tool is rapidly gaining recognition and maturity. Among various advantages over traditional polymerase chain reaction, the ability to visually detect amplification by the incorporation of colorimetric indicators is one of its most unique features. There is an overwhelming variety of LAMP indicators in the literature, yet a comprehensive comparative study is lacking. This study evaluates the use of hydroxynaphthol blue, phenol red, calcein, leuco crystal violet, malachite green, and a fluorescent dye for visual detection. A method for objective quantitative analysis using ImageJ is described that is readily implemented in standard and microfluidic workflows. The work here also includes the largest inter-reader variability study involving 24 participants to evaluate these indicators. We found inaccuracies in visual assessment as bias and/or individual-based perception can exist, solidifying the need for objective analysis. There was not a "universal" indicator, although considerations in sample preparation, storage, and applicability are discussed in length.

A novel loop-mediated isothermal amplification method for identification of four body fluids with smartphone detection.

Messenger RNA profiling for body fluid identification (bfID) is a useful approach to collect contextual information associated with a crime. Current methods require costly fluorescent probes, lengthy amplification protocols and/or time-consuming sample preparation. To simplify this process, we developed a bfID method that has the potential to be rapid in analysis time, inexpensive and fluorescence-free, combining a universal operating procedure with a high-throughout (microwell plate) platform for simultaneous detection of mRNA markers from whole blood, semen, saliva, and vaginal fluid. Full bfID sample preparation and analysis of 23 samples was completed in under 3 h using smart phone optical detection and analysis and show efficacy of the method in a validated blind study. The results provide an efficient, sensitive and specific approach to supplement the current biochemical tests in a forensic laboratory.

Rapid sperm lysis and novel screening approach for human male DNA via colorimetric loop-mediated isothermal amplification.

Correct identification of probative samples is the first crucial step in the analysis of sexual assault kits (SAKs). We report a nucleic acid-based approach, as an alternative to the widely utilized p30 assay, to screening male DNA from SAKs collected from female victims by combining a rapid lysis protocol with an isothermal amplification method. The enzymatic lysis protocol efficiently digests biological material to release nuclear DNA in 10 min in a single closed tube, including resilient cell types such as sperm cells. The amplification and detection of human male specific DNA is achieved through loop-mediated isothermal amplification (LAMP) accompanied with hydroxynaphthol blue, a colorimetric indicator, producing a visually-distinctive color change in the presence of male DNA. The Y-screen approach demonstrated high specificity to human male DNA, can reliably detect target DNA as low as 50 pg, and correctly identified all probative samples from 14 single-blind mock sexual assault samples. In contrast with the widely used p30 assay which requires at least 2 h incubation time and manual application to a lateral flow pad, this Y-screen assay can be completed in half the time, and can be performed in a 96-well format without the need for a fluorescence detector, making facile high-throughput sample screening possible.

microRNA Detection in Blood, Urine, Semen, and Saliva Stains After Compromising Treatments.

Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identification has been the subject of investigation over the past several years. Because of their size and encapsulation within proteins and lipids, miRNAs are inherently less susceptible to degradation than other RNAs. In this work, blood, urine, semen, and saliva were exposed to environmental and chemical conditions mimicking sample compromise at the crime scene. For many treated samples, including 100% of blood samples, miRNAs remained detectable, comparable to the untreated control. Sample degradation varied by body fluid and treatment, with blood remarkably resistant, while semen and saliva are more susceptible to environmental insult. Body fluid identification using relative miRNA expression of blood and semen of the exposed samples was 100% and 94%, respectively. Given the overall robust results herein, the case is strengthened for the use of miRNAs as a molecular method for body fluid identification.

Detection of microRNAs in DNA Extractions for Forensic Biological Source Identification.

Molecular-based approaches for biological source identification are of great interest in the forensic community because of a lack of sensitivity and specificity in current methods. MicroRNAs (miRNAs) have been considered due to their robust nature and tissue specificity; however, analysis requires a separate RNA extraction, requiring an additional step in the forensic analysis workflow. The purpose of this study was to evaluate miRNA detection in blood, semen, and saliva using DNA extraction methods commonly utilized for forensic casework. RT-qPCR analysis revealed that the tested miRNAs were consistently detectable across most tested DNA extraction methods, but detection was significantly reduced compared to RNA extracts in some biological fluids. DNase treatment was not necessary to achieve miRNA-specific results. A previously developed miRNA panel for forensic body fluid identification was evaluated using DNA extracts, and largely demonstrated concordance with results from samples deriving from RNA extracts of semen, blood, and saliva.