Protocols in Current Issue
    COVID-19 Sample Pooling: From RNA Extraction to Quantitative Real-time RT-PCR

    The COVID-19 pandemic requires mass screening to identify those infected for isolation and quarantine. Individually screening large populations for the novel pathogen, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is costly and requires a lot of resources. Sample pooling methods improve the efficiency of mass screening and consume

    A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA
    Authors:  Benno Zehnder, Stephan Urban and Thomas Tu, date: 04/20/2021, view: 550, Q&A: 0

    Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain

    Detection and Quantification of African Swine Fever Virus in MA-104 Cells

    Detection of live African swine fever virus (ASFV) has historically relied on the use of primary swine macrophages (PSM). PSM do not replicate and have to be isolated fresh from donor swine. We previously identified that a MA-104 cells (ATCC #CRL-2378.1), a commercially available cell line isolated from African green monkey (Cercopithecus

    Colorimetric RT-LAMP and LAMP-sequencing for Detecting SARS-CoV-2 RNA in Clinical Samples

    During pandemics, such as the one caused by SARS-CoV-2 coronavirus, simple methods to rapidly test large numbers of people are needed. As a faster and less resource-demanding alternative to detect viral RNA by conventional qPCR, we used reverse transcription loop-mediated isothermal amplification (RT-LAMP). We previously established colorimetric

    A One-enzyme RT-qPCR Assay for SARS-CoV-2, and Procedures for Reagent Production
    Authors:  Sanchita Bhadra, Andre C. Maranhao, Inyup Paik and Andrew D. Ellington, date: 01/20/2021, view: 630, Q&A: 1

    Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed an RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase/DNA polymerase (RTX) (Ellefson

    Evaluation of the Sequence Variability within the PCR Primer/Probe Target Regions of the SARS-CoV-2 Genome
    Authors:  Kashif Aziz Khan and Peter Cheung, date: 12/20/2020, view: 1404, Q&A: 0
    [Abstract] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named 2019-nCoV) is responsible for the recent coronavirus disease (COVID-19) pandemic, and polymerase chain reaction (PCR) is the current standard method for diagnosis from patient samples. As PCR assays are prone to sequence mismatches due to mutations in the viral genome, it ...
    Colorimetric RT-LAMP Methods to Detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)
    [Abstract] Standard diagnostic methods of Coronavirus Disease 2019 (COVID-19) rely on RT-qPCR technique which have limited point-of-care test (POCT) potential due to necessity of dedicated equipment and specialized personnel. LAMP, an isothermal nucleic acid amplification test (NAAT), is a promising technique that may substitute RT-qPCR for POCT of genomic ...
    A Protocol for Simple, Rapid, and Direct Detection of SARS-CoV-2 from clinical samples, using Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP)
    [Abstract] SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all countries was to effectively test the population for the virus. Most countries chose a lockdown ...
    Screening Method for CRISPR/Cas9 Inhibition of a Human DNA Virus: Herpes Simplex Virus
    [Abstract] The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and ...
    Paper Lateral Flow Biosensor for Nodavirus Reverse Transcribed RNA Detection
    Authors:  Dimitra K. Toubanaki and Evdokia Karagouni, date: 08/05/2020, view: 1112, Q&A: 0
    [Abstract] Paper nanobiosensors have been established as an excellent platform for analysis of veterinary and human pathogens causing various diseases. Especially, lateral flow assays or biosensors ideal for sensitive, rapid, robust and accurate analysis in laboratory setups and on-site analysis. Viral RNA detection is of great importance for public health ...

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