Reviewer
Migla Miskinyte
  • Post-Doc, Instituto Gulbenkian de Ciencia
Research fields
  • Molecular Biology, Systems Biology, Bioinformatics, Genetics
Extraction, Purification and Detection of SARS-CoV-2 Nucleic Acid
Author:  Shanghai BioGerm Medical Technology Co. LTD, date: 09/05/2020, view: 2406, Q&A: 2
Nucleic acid extraction and purification kit is used for isolating high-quality pathogen nucleic acids (DNA/RNA) from a variety of specimens. Nucleic acid can be directly extracted from liquid samples such as blood, serum, plasma, urine, nasopharyngeal swabs, and cell culture. And soil samples such as stool, tissue samples need to be pre- treated, obtaining a supernatant and carrying out extraction and purification to obtain the nucleic acids. The obtained nucleic acids can be directly used in related experiments such as Fluorescence quantitative PCR, RT-PCR, biochip analysis, second-generation sequencing.

The SARS-CoV-2 Nucleic Acid Detection Kit is a molecular in vitro diagnostic test that aids in the detection and diagnosis of 2019-nCoV and is based on widely used nucleic acid amplification technology. The product contains oligonucleotide primers and dual-labeled hydrolysis probes and control material used in RT-PCR for the in vitro qualitative detection of SARS-CoV-2 RNA in nasopharyngeal swab, oropharyngeal swab and sputum specimens.

The oligonucleotide primers and probes for detection of SARS-CoV-2 were selected from regions of the virus ORF1ab gene and nucleocapsid (N) gene. The panel is designed for specific detection of the SARS-CoV-2 (two primer/probe sets). An additional primer/probe set to detect the human RNase P gene (RNP) in control samples and clinical specimens is also included in the panel.
Classification of a Massive Number of Viral Genomes and Estimation of Time of Most Recent Common Ancestor (tMRCA) of SARS-CoV-2 Using Phylodynamic Analsysis
Authors:  Xiaowen Hu, Siqin Guan, Yiliang He, Guohui Yi, Lei Yao and Jiaming Zhang, date: 03/20/2024, view: 1202, Q&A: 0

Estimating the time of most recent common ancestor (tMRCA) is important to trace the origin of pathogenic viruses. This analysis is based on the genetic diversity accumulated in a certain time period. There have been thousands of mutant sites occurring in the genomes of SARS-CoV-2 since the COVID-19 pandemic started; six highly linked mutation sites occurred early before the start of the pandemic and can be used to classify the genomes into three main haplotypes. Tracing the origin of those three haplotypes may help to understand the origin of SARS-CoV-2. In this article, we present a complete protocol for the classification of SARS-CoV-2 genomes and calculating tMRCA using Bayesian phylodynamic method. This protocol may also be used in the analysis of other viral genomes.


Key features

• Filtering and alignment of a massive number of viral genomes using custom scripts and ViralMSA.

• Classification of genomes based on highly linked sites using custom scripts.

• Phylodynamic analysis of viral genomes using Bayesian evolutionary analysis sampling trees (BEAST).

• Visualization of posterior distribution of tMRCA using Tracer.v1.7.2.

• Optimized for the SARS-CoV-2.


Graphical overview



Graphical workflow of time of most recent common ancestor (tMRCA) estimation process

Dual-target Bridging ELISA for Bispecific Antibodies
Authors:  Min Pei, Yao Wang, Lei Tang, Weitao Wu, Chunhe Wang and Yi-Li Chen, date: 10/05/2022, view: 1873, Q&A: 0

Bispecific antibodies (BsAbs) are typically monoclonal antibody (mAb)–derived molecular entities engineered to bind to two distinct targets, including two antigens or two epitopes on the same antigen. When compared to parental monoclonal antibodies or combinational therapies, the generated BsAbs have the ability to bridge the two targets and thus may offer additional clinical benefits. Characterizing BsAbs’ ability to bind to both targets simultaneously is critical for their biotherapeutic development. A range of bi-functional quantitative bridging assays to enable target-specific capture and detection of binding properties include enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and cell-based flow cytometry. Developing suitable and robust cell-based bioassays is more challenging than non-cell-based binding assays because cell-based assays with complex matrices can be inherently variable and often lack precision. Compared to SPR, ELISA has a rapid setup and readily available method, being widely and extensively applied in almost every laboratory. Here, we describe a dual-target bridging ELISA assay that characterizes the ability of a HER2(human epidermal growth factor receptor 2)/PD-L1(programmed cell death ligand 1) BsAb in binding to both HER2 and PD-L1 simultaneously, a prerequisite for its envisioned mode of action.


Graphical abstract:




Activity-based Crosslinking to Identify Substrates of Thioredoxin-domain Proteins in Malaria Parasites
Authors:  David W. Cobb, Grace S. Woods and Vasant Muralidharan, date: 02/20/2022, view: 1783, Q&A: 0

Malaria remains a major public health issue, infecting nearly 220 million people every year. The spread of drug-resistant strains of Plasmodium falciparum around the world threatens the progress made against this disease. Therefore, identifying druggable and essential pathways in P. falciparum parasites remains a major area of research. One poorly understood area of parasite biology is the formation of disulfide bonds, which is an essential requirement for the folding of numerous proteins. Specialized chaperones with thioredoxin (Trx) domains catalyze the redox functions necessary for breaking incorrect and forming correct disulfide bonds in proteins. Defining the substrates of these redox chaperones is difficult and immunoprecipitation based assays cannot distinguish between substrates and interacting partners. Further, the substrate or client interactions with the redox chaperones are usually transient in nature. Activity based crosslinkers that rely on the nucleophilic cysteines on Trx domains and the disulfide bond forming cysteines on clients provide an easily scalable method to trap and identify the substrates of Trx-domain containing chaperones. The cell permeable crosslinker divinyl sulfone (DVSF) is active only in the presence of nucleophilic cysteines in proteins and, therefore, traps Trx domains with their substrates, as they form mixed disulfide bonds during the course of their catalytic activity. This allows the identification of substrates that rely on Trx activity for their folding, as well as discovering small molecules that interfere with Trx domain activity.


Graphic abstract:



Identification of thioredoxin domain substrates via divinylsulfone crosslinking and immunoprecipitation-mass spectrometry.

A Standard Operative Procedure for Safe-handling of Remains and Wastes of COVID-19 Patients
The ongoing coronavirus disease-2019 (COVID-19) pandemic has raised significant public health issues which need to be attended. To enable efficient handling of the situation and prevent the spread of the epidemic, healthcare professionals require various standard operating procedures (SOPs). Emerging evidence suggests high infectivity of the novel coronavirus strain–severe acute respiratory syndrome virus-2 (SARS-CoV-2)–causing COVID-19. The remains and wastes of COVID-19 patients can be a potential source of the virus exposure to the healthcare professionals if not handled with adequate precaution. Various institutions have issued SOPs in this regard, but a comprehensive approach is missing, which creates difficulty in interpretation and application of these SOPs. We have developed a comprehensive protocol for disposal of remains and wastes of the COVID-19 patients.We are following this protocol at our institution without any untoward event until date.
Environmental Conditioning and Aerosol Infection of Mice
Authors:  Eriko Kudo and Akiko Iwasaki, date: 04/20/2020, view: 3985, Q&A: 0
Influenza infection models in mice are widely used to study flu-mediated immune responses and pathology. However, most laboratory mice are housed at 20 °C and 50% relative humidity (RH). To better recapitulate influenza epidemics and immune responses during winter seasons, mice were housed at 20 °C under different humidity conditions, 10-20% or 50% RH. Here, we describe a protocol for using aerosolized droplets to infect mice with influenza under different environmental conditions. Using this method enables influenza infection studies performed under more physiologically relevant conditions which better mimics human viral exposure.
Eimeria vermiformis Infection Model of Murine Small Intestine
Authors:  Patrícia Figueiredo-Campos, Cristina Ferreira, Birte Blankenhaus and Marc Veldhoen, date: 12/20/2018, view: 5030, Q&A: 0
Eimeria vermiformis is a tissue specific, intracellular protozoan that infects the murine small intestinal epithelia, which has been widely used as a coccidian model to study mucosal immunology. This mouse infection model is valuable to investigate the mechanisms of host protection against primary and secondary infection in the small intestine. Here, we describe the generation of an E. vermiformis stock solution, preparation of sporulated E. vermiformis to infect mice and determination of oocysts burden. This protocol should help to establish a highly reproducible natural infection challenge model to study immunity in the small intestine. The information obtained from using this mouse model can reveal fundamental mechanisms of interaction between the pathogen and the immune response, e.g., provided by intraepithelial lymphocytes (IEL) at the basolateral site of epithelial cells but also a variety of other immune cell populations present in the gut.
H1N1 Virus Production and Infection
Authors:  Binbin Zhao, Jiaoyu Shan, Rui Xiong, Ke Xu and Bin Li, date: 10/20/2018, view: 5871, Q&A: 0
Influenza A virus is a member of orthomyxoviridae family causing wide-spread infections in human respiratory tract. Mouse infection model is widely used in antiviral research and pathogenesis study against influenza A virus. Here, we report a protocol in infected mice with different virus doses and strains to explore how an inhibitor of lysine-specific demethylase (LSD1) impacts disease progression.
Quantification of Bacterial Attachment to Tissue Sections
Authors:  Batya Isaacson, Tehila Hadad, Gilad Bachrach and Ofer Mandelboim, date: 03/05/2018, view: 7394, Q&A: 0
Here we describe a method to test bacterial adhesion to paraffin embedded tissue sections. This method allows examining binding of different bacterial strains, transfected with a fluorescent protein reporter plasmid to various tissues, to better understand different mechanisms such as colonization. This assay provides a more physiological context to bacterial binding, than would have been achieved using adhesion assays to cell lines. The sections can be imaged using fluorescent microscopy and adhesion of various bacterial strains can be quantified and tested, simultaneously.
Analysis of the Virulence of Uropathogenic Escherichia coli Strain CFT073 in the Murine Urinary Tract
Authors:  Anna Waldhuber, Manoj Puthia, Andreas Wieser, Catharina Svanborg and Thomas Miethke , date: 02/05/2017, view: 10422, Q&A: 0
This urinary tract infection model was used to monitor the efficacy of a new virulence factor of the uropathogenic Escherichia coli strain CFT073 in vivo. The new virulence factor which we designated TIR-containing protein C (TcpC) blocks Toll-like receptor signaling and the NLRP3 inflammasome signaling cascade by interacting with key components of both pattern recognition receptor systems (Cirl et al., 2008; Waldhuber et al., 2016). We infected wild type and knock-out mice with wildtype CFT073 and a mutant CFT073 strain lacking tcpC. This protocol describes how the mice were infected, how CFT073 was prepared and how the infection was monitored. The protocol was derived from our previously published work and allowed us to demonstrate that TcpC is a powerful virulence factor by increasing the bacterial burden of CFT073 in the urine and kidneys. Moreover, TcpC was responsible for the development of kidney abscesses since infection of mice with wildtype but not tcpC-deficient CFT073 mutants caused this complication.
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