COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme.

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K/Q/A, and N501Y mutations, at least one of which is found in nearly all major variants. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 261 clinical samples yielded a SNP concordance of 97.3% and agreement of 98.9% (258 of 261) for SARS-CoV-2 lineage classification, using SARS-CoV-2 whole-genome sequencing and/or real-time RT-PCR as test comparators. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR as a faster and simpler diagnostic method compared with sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.

High-Yielding Separation and Collection of Plasma from Whole Blood Using Passive Filtration.

Lateral flow tests and hand-held analyzers facilitate diagnostic testing in resource limited settings and at the point-of-care. However, many of these devices require sample preparation such as plasma separation to remove cells and isolate the liquid portion of blood. Specifically, the separation of plasma from blood is necessary for routine health assessments such as comprehensive metabolic panels and chronic HIV viral load monitoring. Away from laboratories, this type of processing has been addressed by unconventional, hand-operated centrifuge devices (high volume) or plasma separation membranes (PSM) coupled with lateral flow tests (low volume). Herein, we describe a device that separates and stores plasma from undiluted blood using only passive filtration in less than 10 min. Integrating a PSM with a prefilter and absorbent material yields a 3-fold increase in separation efficiency compared to similar devices using passive filtration. We demonstrate the reproducibility of our device across the physiological range of hematocrits (20-50%) with an average recovered plasma volume of 61.7 ± 2.6 µL. Maximum separation efficiency (53.8%, 65.6 ± 3.9 µL plasma) was achieved for a sample of whole blood (30% hematocrit) in 10 min. We evaluate the purity of our plasma sample by quantitation of hemoglobin and report hemolysis as either minimal (≤5%) or undetectable (≤1%). Specific recovery of human IgG, IFN-γ, and HIV-1 RNA indicate the diagnostic utility of plasma obtained from our device is unchanged compared to plasma obtained via centrifugation. Finally, we demonstrate the use of recovered plasma, applied via "stamping", to successfully conduct a commercial lateral flow immunochromatographic assay for tetanus antibodies. This device platform is capable of producing pure plasma samples from blood to facilitate tests in resource limited settings to improve access to healthcare.

Synergistic use of electroosmotic flow and magnetic forces for nucleic acid extraction.

Nucleic acid sample preparation is essential for biological sample-based diagnostics. It is crucial that diagnostic tests be both specific and sensitive as to provide the most accurate diagnosis possible. Inefficient sample preparation can hinder the specificity and sensitivity of these tests since carryover contaminants can inhibit downstream processes, such as amplification. Microfluidic devices have been used previously to extract nucleic acids from a biological sample due to lower reagent volumes and ease of use. A novel microfluidic chip has been designed for nucleic acid sample preparation which combines electroosmotic flow and magnetic bead-based extraction to isolate DNA from a plasma sample. A steady electric field was incorporated into the microfluidic chip design, which when combined with a glass clover slip and a voltage differential, creates electroosmotic flow. With the goal of isolating nucleic acids into a clean, inhibitor free solution, the electroosmotic flow is the driving force and separation mechanism purifying the DNA sample captured on magnetic beads in the microfluidic chip system. Carryover volume, or the volume of unwanted sample contaminants that accompany the nucleic acids into the final elution buffer, was minimized to 0.22 ± 0.03%. In combination with magnetic bead based nucleic acid extraction techniques, a 15% increase in DNA extraction yield is reported for the microfluidic chip with the voltage applied versus without. Although the literature on nucleic acid separation in microfluidic chips is abundant, this is the first to combine microfluidic chip design, magnetic bead-based isolation and electroosmotic flow.

Vortex- and Centrifugation-Free Extraction of HIV-1 RNA.

BACKGROUND AND OBJECTIVE: HIV viral load measurements play a critical role in monitoring disease progression in those who are on antiretroviral treatment. In order to obtain an accurate measurement, rapid sample preparation techniques are required. There is an unmet need for HIV extraction instruments in resource-limited settings, where HIV prevalence is high. Therefore, the objective of our study was to develop a three-dimensional (3D) microfluidic system to extract HIV-1 RNA with minimal electricity and without complex laboratory instruments. METHODS: A 3D microfluidic system was designed in which magnetic beads bound with nucleic acids move through immiscible oil-water interfaces to separate HIV-1 RNA from the sample. Polymerase chain reaction (PCR) amplification was used to quantify the total amount of HIV-1 RNA extracted as we optimized the system through chip design, bead type, carry-over volume, carrier RNA concentration, and elution buffer temperature. Additionally, the extraction efficiency of the 3D microfluidic system was evaluated by comparing with a Qiagen EZ1 Advanced XL instrument using 20 HIV-1-positive plasma samples. RESULTS: Our method has near-perfect (100%) extraction efficiency in spiked serum samples with as little as 50 copies/mL starting sample. Furthermore, we report carry-over volumes of 0.31% ± 0.006% of total sample volume. Using the EZ1 Advanced XL as a gold standard, the average percentage HIV-1 RNA extracted using the microchip was observed to be 65.4% ± 24.6%. CONCLUSIONS: From a clinical perspective, the success of our method opens up its possible use in diagnostic tests for HIV in the remote areas where access to vortexes and centrifuges is not available. Here we present a proof-of-concept device which, with further development, could be used for sample preparation at the point of care.

Multiplexed, Patterned-Paper Immunoassay for Detection of Malaria and Dengue Fever.

Multiplex assays detect the presence of more than one analyte in a sample. For diagnostic applications, multiplexed tests save healthcare providers time and resources by performing many assays in parallel, minimizing the amount of sample needed and improving the quality of information acquired regarding the health status of a patient. These advantages are of particular importance for those diseases that present with general, overlapping symptoms, which makes presumptive treatments inaccurate and may put the patient at risk. For example, malaria and dengue fever are febrile illnesses transmitted through mosquito bites, and these common features make it difficult to obtain an accurate diagnosis by symptoms alone. In this manuscript, we describe the development of a multiplexed, patterned paper immunoassay for the detection of biomarkers of malaria and dengue fever: malaria HRP2, malaria pLDH, and dengue NS1 type 2. In areas coendemic for malaria and dengue fever, this assay could be used as a rapid, point-of-care diagnostic to determine the cause of a fever of unknown origin. The reagents required for each paper-based immunoassay are separated spatially within a three-dimensional device architecture, which allows the experimental conditions to be adjusted independently for each assay. We demonstrate the analytical performances of paper-based assays for each biomarker and we show that there is no significant difference in performance between the multiplexed immunoassay and those immunoassays performed in singleplex. Additionally, we spiked individual analytes into lysed human blood to demonstrate specificity in a clinically relevant sample matrix. Our results suggest multiplex paper-based devices can be an essential component of diagnostic assays used at the point-of-care.

Adsorption and isolation of nucleic acids on cellulose magnetic beads using a three-dimensional printed microfluidic chip.

While advances in genomics have enabled sensitive and highly parallel detection of nucleic acid targets, the isolation and extraction of the nucleic acids remain a critical bottleneck in the workflow. We present here a simple 3D printed microfluidic chip that allows for the vortex and centrifugation free extraction of nucleic acids. This novel microfluidic chip utilizes the presence of a water and oil interface to filter out the lysate contaminants. The pure nucleic acids, while bound on cellulose particles, are magnetically moved across the oil layer. We demonstrated efficient and rapid extraction of spiked Human Papillomavirus (HPV) 18 plasmids in specimen transport medium, in under 15 min. An overall extraction efficiency of 61% is observed across a range of HPV plasmid concentrations (5 × 10(1) to 5 × 10(6) copies/100 µl). The magnetic, interfacial, and viscous drag forces inside the microgeometries of the chip are modeled. We have also developed a kinetics model for the adsorption of nucleic acids on cellulose functionalized superparamagnetic beads. We also clarify here the role of carrier nucleic acids in the adsorption and isolation of nucleic acids. Based on the various mechanistic insights detailed here, customized microfluidic devices can be designed to meet the range of current and emerging point of care diagnostics needs.

A device architecture for three-dimensional, patterned paper immunoassays.

Diagnostic assays can provide valuable information about the health status of a patient, which include detection of biomarkers that indicate the presence of an infection, the progression or regression of a disease, and the efficacy of a course of treatment. Critical healthcare decisions must often be made at the point-of-care, far from the infrastructure and diagnostic capabilities of centralized laboratories. There exists an obvious need for diagnostic tools that are designed to address the unique challenges encountered by healthcare workers in limited-resource settings. Paper, a readily-available and inexpensive commodity, is an attractive medium with which to develop diagnostic assays for use in limited-resource settings. In this article, we describe a device architecture to perform immunoassays in patterned paper. These paper-based devices use a combination of lateral and vertical flow to control the wicking of fluid in three-dimensions. We provide guidelines to aid in the design of these devices and we illustrate how patterning can be used to tune the duration and performance of the assay. We demonstrate the use of these paper-based devices by developing a sandwich immunoassay for human chorionic gonadotropin (hCG) in urine, a biomarker of pregnancy. We then directly compare the qualitative and quantitative results of these paper-based immunoassays to commercially available lateral flow tests (i.e., the home pregnancy test). Our results suggest paper-based devices may find broad utility in the development of immunoassays for use at the point-of-care.