Vigilant: An Engineered VirD2-Cas9 Complex for Lateral Flow Assay-Based Detection of SARS-CoV2.
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ABSTRACT:
SUBMITTER: Marsic T
PROVIDER: S-EPMC8056947 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Since the onset of the SARS-CoV-2 pandemic, several COVID-19 detection methods, both commercially available and in the lab, have been developed using different biomolecules as analytes and different detection and sampling methods with high analytical performance. Developing novel COVID-19 detection assays is an exciting research field, as rapid accurate diagnosis is a valuable tool to control the current pandemic, and also because the acquired knowledge can be deployed for facing future infectious outbreaks. We here developed a novel gold-nanoparticle-based nucleic acid lateral flow assay for the rapid, visual, and quantitative detection of SARS-CoV-2. Our method was based on the use of a DNA internal standard (competitor) for quantification and involved RT-PCR, the hybridization of biotinylated PCR products to specific oligonucleotide probes, and detection with a dual lateral flow assay using gold nanoparticles conjugated to an anti-biotin antibody as reporters. The developed test allowed for rapid detection by the naked eye and the simultaneous quantification of SARS-CoV-2 in nasopharyngeal swabs with high specificity, detectability, and repeatability. This novel molecular strip test for COVID-19 detection represents a simple, cost-effective, and accurate rapid test that is very promising to be used as a future diagnostic tool.
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Project description:This paper proposes that the signal intensity of a lateral flow assay (LFA) strip can be increased by pressing the top of the strip, effectively reducing its flow rate. The reduced flow rate allows more time for antigen-antibody interactions to occur, resulting in increased signal intensity and an improved detection limit. To assess the potential of the pressed LFA (pLFA) strip, C-reactive protein (CRP) diluted in phosphate-buffered saline (PBS) and serum is detected, affording signal enhancement and a lowered limit of detection. Additionally, to show that the signal enhancement by pressure-induced flow delay applies to existing LFA products, commercially available COVID-19 antigen test strips are pressed, and signal enhancement is observed. Lastly, we show that the signal intensity of COVID-19 LFA kits can be increased by approximately two-fold at maximum by applying pressure on top of the manufactured product. This study suggests that pressed LFA strips can be used to reduce the chances of determining ambiguous signals as false-negative results and can potentially improve the detection sensitivity.Graphical abstractSupplementary informationThe online version contains supplementary material available at 10.1007/s13206-022-00085-w.
| S-EPMC9607699 | biostudies-literature
Project description:Inexpensive, simple, rapid diagnostics are necessary for efficient detection, treatment, and mitigation of COVID-19. Assays for SARS-CoV2 using reverse transcription polymerase chain reaction (RT-PCR) offer good sensitivity and excellent specificity, but are expensive, slowed by transport to centralized testing laboratories, and often unavailable. Antigen-based assays are inexpensive and can be rapidly mass-produced and deployed at point-of-care, with lateral flow assays (LFAs) being the most common format. While various manufacturers have produced commercially available SARS-Cov2 antigen LFAs, access to validated tests remains difficult or cost prohibitive in low-and middle-income countries. Herein, we present a visually read open-access LFA (OA-LFA) using commercially-available antibodies and materials for the detection of SARS-CoV-2. The LFA yielded a Limit of Detection (LOD) of 4 TCID50/swab of gamma irradiated SARS-CoV-2 virus, meeting the acceptable analytical sensitivity outlined by in World Health Organization target product profile. The open-source architecture presented in this manuscript provides a template for manufacturers around the globe to rapidly design a SARS-CoV2 antigen test.
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Project description:BackgroundThe SARS-CoV-2 outbreak has emerged at the end of 2019. Aside from the detection of viral genome with specific RT-PCR, there is a growing need for reliable determination of the serological status. We aimed at evaluating five SARS-CoV-2 serology assays.MethodsAn in-house immunofluorescence assay (IFA), two ELISA kits (EUROIMMUN® ELISA SARS-CoV-2 IgG and NovaLisa® SARS-CoV-2 IgG and IgM) and two lateral flow assays (T-Tek® SARS-CoV-2 IgG/IgM Antibody Test Kit and Sure Bio-tech® SARS-CoV-2 IgM/IgG Antibody Rapid Test) were compared on 40 serums from RT-PCR-confirmed SARS-CoV-2 infected patients and 10 SARS-CoV-2 RT-PCR negative subjects as controls.ResultsControl subjects tested negative for SARS-CoV-2 antibodies with all five systems. Estimated sensitivities varied from 35.5 to 71.0% for IgG detection and from 19.4 to 64.5% for IgM detection. For IgG, in-house IFA, EuroImmun, T-Tek and NovaLisa displayed 50-72.5% agreement with other systems except IFA vs EuroImmun and T-Tek vs NovaLisa. Intermethod agreement for IgM determination was between 30 and 72.5%.DiscussionThe overall intermethod agreement was moderate. This inconsistency could be explained by the diversity of assay methods, antigens used and immunoglobulin isotype tested. Estimated sensitivities were low, highlighting the limited value of antibody detection in CoVID-19.ConclusionComparison of five systems for SARS-CoV-2 IgG and IgM antibodies showed limited sensitivity and overall concordance. The place and indications of serological status assessment with currently available tools in the CoVID-19 pandemic need further evaluations.
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Project description:The COVID-19 pandemic is among the greatest health and socioeconomic crises in recent history. Although COVID-19 vaccines are being distributed, there remains a need for rapid testing to limit viral spread from infected individuals. We previously identified the SARS-CoV-2 spike protein N-terminal domain (NTD) binding DNA aptamer 1 (SNAP1) for detection of SARS-CoV-2 virus by aptamer-antibody sandwich enzyme-linked immunoassay (ELISA) and lateral flow assay (LFA). In this work, we identify a new aptamer that also binds at the NTD, named SARS-CoV-2 spike protein NTD-binding DNA aptamer 4 (SNAP4). SNAP4 binds with high affinity (<30 nM) for the SARS-CoV-2 spike protein, a 2-fold improvement over SNAP1. Furthermore, we utilized both SNAP1 and SNAP4 in an aptamer sandwich LFA (AptaFlow), which detected SARS-CoV-2 UV-inactivated virus at concentrations as low as 106 copies/mL. AptaFlow costs <$1 per test to produce, provides results in <1 h, and detects SARS-CoV-2 at concentrations that indicate higher viral loads and a high probability of contagious transmission. AptaFlow is a potential approach for a low-cost, convenient antigen test to aid the control of the COVID-19 pandemic.
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Project description:BackgroundWhile the diagnosis of SARS-CoV-2 infection is primarily based on detection of viral RNA, the detection of SARS-CoV-2 antibodies is useful for assessing past prevalence of the disease, and in corroborating a current infection in challenging cases. Sensitive and specific immunoassays provide the ability to identify exposure to SARS-CoV-2, to determine seroconversion, to confirm eligibility for donation of convalescent plasma as well as play an essential part in epidemiological studies. We report on the validation of the Ansh Laboratories SARS-CoV-2 IgG and SARS-CoV-2 IgM ELISA immunoassays. These assays were evaluated for detection of anti-SARS-CoV-2 IgG and IgM antibodies for clinical use in our hospital as part of an orthogonal testing algorithm recommended by the CDC.MethodsDiagnostic specificity and sensitivity of the IgG and IgM ELISA assays were tested using samples confirmed to be negative or positive for COVID-19 by RT-PCR. We also evaluated precision, analytical interference, and cross-reactivity with known cases of infection with other viruses. Additionally, we validated concordance with molecular and other serological testing and evaluated seroconversion in our patient population.ResultsThe IgG and IgM ELISA assays showed acceptable precision, were robust to analytical interference and did not exhibit cross reactivity with specimens positive for common respiratory viruses. Both assays exhibited 95% agreement with a primary screening serological assay utilized at our institution as well as with a reference laboratory semi-quantitative method. Concordance with RT-PCR was excellent > 6 days after symptom onset (100%).ConclusionsThe Ansh SARS-CoV-2 ELISA assays have good analytical performance suitable for clinical use.
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Project description:Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings.
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Project description:BackgroundAs vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing.MethodsWe have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques.FindingsWe show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test.InterpretationTaken together, the COVID-19 NAb-testTM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care.FundingSupport was received from the Victorian Operational Infrastructure Support Program and the Australian Government Department of Health. This work was supported by grants from the Department of Health and Human Services of the Victorian State Government; the ARC (CE140100011, CE140100036), the NHMRC (1113293, 2002317 and 1116530), and Medical Research Future Fund Awards (2005544, 2002073, 2002132). Individual researchers were supported by an NHMRC Emerging Leadership Level 1 Investigator Grants (1194036), NHMRC APPRISE Research Fellowship (1116530), NHMRC Leadership Investigator Grant (1173871), NHMRC Principal Research Fellowship (1137285), NHMRC Investigator Grants (1177174 and 1174555) and NHMRC Senior Principal Research Fellowships (1117766 and 1136322). Grateful support was also received from the A2 Milk Company and the Jack Ma Foundation.
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Project description:Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially available reagents into a CRISPR/Cas9-based lateral flow assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences with single-base specificity. This approach requires minimal equipment and represents a simplified platform for field-based deployment. We also developed a rapid, multiplex fluorescence CRISPR/Cas9 nuclease cleavage assay capable of detecting and differentiating SARS-CoV-2, influenza A and B, and respiratory syncytial virus in a single reaction. Our findings provide proof-of-principle for CRISPR/Cas9 point-of-care diagnosis as well as a scalable fluorescent platform for identifying respiratory viral pathogens with overlapping symptomology.
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Project description:Identification of the main SARS-CoV-2 variants in real time is of interest to control the virus and to rapidly devise appropriate public health responses. The RT-qPCR is currently considered to be the reference method to screen SARS-CoV-2 mutations, but it has some limitations. The multiplexing capability is limited when the number of markers to detect increases. Moreover, the performance of this allele-specific method may be impacted in the presence of new mutations. Herein, we present a proof-of-concept study of a simple molecular assay to detect key SARS-CoV-2 mutations. The innovative features of the assay are the multiplex asymmetric one-step RT-PCR amplification covering different regions of SARS-CoV-2 S gene and the visual detection of mutations on a lateral flow DNA microarray. Three kits (Kit 1: N501Y, E484K; Kit 2: L452R, E484K/Q; Kit 3: K417N, L452R, E484K/Q/A) were developed to match recommendations for surveillance of SARS-CoV-2 variants between January and December 2021. The clinical performance was assessed using RNA extracts from 113 SARS-CoV-2-positive samples with cycle thresholds <30, and results demonstrated that our assay allows specific and sensitive detection of mutations, with a performance comparable to that of RT-qPCR. The VAR-CoV assay detected four SARS-CoV-2 targets and achieved specific and sensitive screening of spike mutations associated with the main variants of concern, with a performance comparable to that of RT-qPCR. With well-defined virus sequences, this assay can be rapidly adapted to other emerging mutations; it is a promising tool for variant surveillance.
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