Designing a PCR test for Coronavirus outbreak (SARS-CoV-2)

In light of the recent emergence of the SARS-CoV-2 worldwide, accurate and novel detection methods for the diagnosis of the disease are mandatory.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), previously known by the provisional name 2019 novel coronavirus (2019-nCoV), is a positive-sense single-stranded RNA virus. It is contagious in humans and is the cause of the ongoing pandemic of coronavirus disease 2019 (COVID-19) that has been designated a Public Health Emergency of International Concern by the World Health Organization (WHO).

SARS-CoV-2 has close genetic similarity to bat coronaviruses, from which it likely originated. An intermediate animal reservoir such as a pangolin is also thought to be involved in its introduction to humans. From a taxonomic perspective, SARS-CoV-2 is classified as a strain of the species severe acute respiratory syndrome-related coronavirus (SARSr-CoV).

Because the strain was first discovered in Wuhan, China, it has sometimes been referred to as the «Wuhan virus» or «Wuhan coronavirus». To avoid confusion with the disease SARS, the WHO sometimes refers to the virus as «the virus responsible for COVID-19» or «the COVID-19 virus» in public health communications. Both the virus and the disease are often called «coronavirus» by the general public, but scientists and most journalists typically use more precise terms.

Phylogenetics and taxonomy

SARS-CoV-2 belongs to the broad family of viruses known as coronaviruses. It is a positive-sense single-stranded RNA (+ssRNA) virus. Other coronaviruses are capable of causing illnesses ranging from the common cold to more severe diseases such as Middle East respiratory syndrome (MERS).

Like the SARS-related coronavirus strain implicated in the 2003 SARS outbreak, SARS-CoV-2 is a member of the subgenus Sarbecovirus.  Its RNA sequence is approximately 30,000 bases in length. SARS-CoV-2 is unique among known betacoronaviruses in its incorporation of a polybasic cleavage site, a characteristic known to increase pathogenicity and transmissibility in other viruses.

Structural biology

Illustration of Coronavirus virion

Each SARS-CoV-2 virion is approximately 50–200 nanometres in diameter. Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. The spike protein, which has been imaged at the atomic level using cryogenic electron microscopy, is the protein responsible for allowing the virus to attach to the membrane of a host cell.

Protein modeling experiments on the spike protein of the virus soon suggested that SARS-CoV-2 has sufficient affinity to the angiotensin converting enzyme 2 (ACE2) receptors of human cells to use them as a mechanism of cell entry.  By 22 January 2020, a group in China demonstrated that ACE2 could act as the receptor for SARS-CoV-2. Initial spike protein priming by transmembrane protease, serine 2 (TMPRSS2) is essential for entry of SARS-CoV-2. After a SARS-CoV-2 virion attaches to a target cell, the cell’s protease TMPRSS2 cuts open the spike protein of the virus, exposing a fusion peptide. The virion then releases RNA into the cell, forcing the cell to produce copies of the virus that are disseminated to infect more cells. SARS-CoV-2 produces at least three virulence factors that promote shedding of new virions from host cells and inhibit immune response.

COVID-19 is an emerging, rapidly evolving situation.
Get the latest public health information from CDC: https://www.coronavirus.gov .
Get the latest research from NIH: https://www.nih.gov/coronavirus.

Laboratory testing for 2019 novel coronavirus (2019-nCoV) in suspected human cases

Accordingly to the Who recommendation «Routine confirmation of cases of COVID-19 is based on detection of unique sequences of virus RNA by NAAT such as real-time reverse-transcription polymerase chain reaction (rRT-PCR) with confirmation by nucleic acid sequencing when necessary. The viral genes targeted so far include the N, E, S and RdRP genes. Examples of protocols used may be found here. RNA extraction should be done in a biosafety cabinet in a BSL-2 or equivalent facility. Heat treatment of samples before RNA extraction is not recommended»

Content instructions for deliverable 2

1. Describe the method recommended by the Who for the detection of COVID 19.

2. Download the genome of the COVID 19 (available in GenBank, NCBI genome ID MN908947).

3. Open the FASTA format

2. Design a primer set contained from the virus sequences recommended above, in order to provide a PCR product. Make an scheme signaling the position of the primers

General instructions for deliverable 2

  • The file must be in pdf format, and the name of the file as «DELIVERABLE 2 NIU«. This is very important to organize the files.
  • The deadline for the telematic delivering is 13/04/2020 12:00 PM.
  • It is mandatory to be logged in the website in order to get the receipt.
  • Finally, I will post the answer here as a feedback.

After being logged for identification, the file must be upload here:

Upload File (50 MB max)




Further bibliography

SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) Sequences

The tables below list the SARS-CoV-2 sequences currently available in GenBank and the Sequence Read Archive (SRA).

The sequence lists were last updated Friday Mar 18 14:25 2020 EST, and are updated as additional sequences are released. The table content is available for download.

PubMed Articles

Articles in PubMed

Nucleotide Sequences

You can view and download these 174 GenBank sequences and 1 RefSeq sequence in Entrez Nucleotide and the new NCBI Virus resource.

Genomic organisation of isolate Wuhan-Hu-1, the earliest sequenced sample of SARS-CoV-2
NCBI genome IDMN908947
Genome size29,903 bases
Year of completion2020

BLAST against Betacoronavirus sequences

ClinicalTrials.gov

View clinical studies listed on ClinicalTrials.gov.

4 comentarios sobre “Designing a PCR test for Coronavirus outbreak (SARS-CoV-2)

  1. Paula Alcalá Pavo dice:

    Hola Isabel!
    En el último apartado de la entrega, donde tenemos que diseñar unos primers, nos indica que lo hagamos a partir de las secuencias del virus recomendadas anteriormente. ¿A cuáles se refiere?

    • Isabel dice:

      Hola, Paula,
      se refiere a la secuencia que obtendrás de estes link, que indica el enunciado más arriba
      . Download the genome of the COVID 19 (available in GenBank, NCBI genome ID MN908947).

      • Paula Alcalá Pavo dice:

        Gracias Isabel!

        Por otro lado, ¿podemos escoger cualquier fragmento de 100pb del ADN para amplificar que sea específico del COVID-19?

        ¿Para comprobar las estructuras secundarias en el link que nos proporcionaste de ThermoFisher, ponemos un valor para detectar la sensibilidad de los dímeros de 1 o, el que sale por defecto (3)?

        • Isabel dice:

          Hola, Paula,
          he quitado del enunciado la restricción de los 100 pb. Antes de realizar el diseño de la PCR, espera a los problemas de mañana miércoles que explicaré herramientas informáticas para seleccionar los primers.
          saludos!

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