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Coronaviruses are a family of viruses, which can cause serious pathologies such as pneumonia-like disease. The new coronavirus first reported in WuhanHubei provinceChina, since mid-December 2019 is now known as the Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) which causes a disease named COVID-19 by the World Health Organization (WHO), acronym for COronaVIrus Disease 2019. The global situation of COVID-19 has been described by WHO as a pandemic after being spread worldwide. The SARS-CoV-2 is affecting 212 countries and territories around the world.

SARS-CoV-2 is the seventh coronavirus known.to.infect.humans, as Middle.East respiratory.syndrome (MERS-Cov) and SARS-Cov SARS-CoV-2 causes respiratory.and intestinal.infections however HKU1, NL63, OC43 and 229E are associated with mild symptoms (Andersen et al. 2020). SARS-Cov first emerged in the Guangdong province, China (2002 - 2003 epidemic). It affected around 8.000 individuals in more than 30 countries and has caused the death of 800 patients (Du Toit, 2020). Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by mortality in the third of patients. dromedaries are reported as an important source of infection. SARS and MERS coronaviruses likely originated in bats and have been transmitted directly to humans from market civets and dromedary camels (Haverkamp et al. 2018).

The COVID-19 pandemic has required border closedown, city lockdown, closure of schools, universities and laboratories in multiple countries. In Algeria the first cases were reported on March 1st in Blida province, near Algiers (http://covid19.sante.gov.dz/carte).

Main-structure-of-coronaviruses


Image credit: https://www.freepik.com/


 

 

The life cycle of coronavirus in host cell
This is a simplified illustration of the coronavirus cycle!
For more molecular details, please refer to the articles cited below and other related papers.

Abbreviations

  • ACE2: Angiotensin Converting Enzyme 2
  • ERGIC: ER-Golgi Intermediate Compartment
  • ER: Endoplasmic reticulum

References

  • Adnan Shereen, M., Khan, S., Kazmi, A., Bashir, N., & Siddique, R. (2020). COVID-19 infection: origin, transmission, and characteristics of human coronaviruses. Journal of Advanced Research.
  • de Wit, E., van Doremalen, N., Falzarano, D. et al. SARS and MERS: recent insights into emerging coronaviruses. Nat Rev Microbiol 14, 523–534 (2016).
  • Matthew B. Frieman and Stuart Weston, Univ. of Maryland School of Medicine; Fields Virology; Fenner and White’s Medical Virology; Nature; Science; The Lancet; New England Journal of Medicine; Centers for Disease Control and Prevention.
  • Peiris, M., Anderson, L. and Osterhaus, A., 2008. Severe Acute Respiratory Syndrome. Chichester: John Wiley & Sons.

 

Detection of SARS-CoV-2 using qRT-PCR
1- Collect specimens (e.g. Nasopharyngeal swab)
2- Preserve specimens in the Viral Transport Medium (VTM)
3- Extract the viral RNA using a Viral RNA Mini Kit.
4- Prepare the qRT-PCR Mix (e.g. Superscript III one step RT-PCR).
5- Set the RT-PCR program and launch the reaction:

    • Reverse transcription: 50 °C for 15 min (01 cycle)
    • Denaturation: 95 °C for 3 min (01 cycle)
    • Amplification: 95 °C for 15 s, 58 °C for 30 s (40-50 cycles)
    • Cooling: 40 °C for 30 s and then 4 °C (∞).

 qPCR Principle

In quantitative PCR we usually use a specific fluorescent probe in addition to primers in order to detect the amplification of the target gene. The probe sequence is labeled at the 5’ end with a fluorescent reporter dye (F) and at the 3’ end with a quencher dye (Q). In case the probe does not anneal to a specific sequence, the fluorescent reporter will stay physically close to the quencher which will cause the quenching of the reporter’s fluorescence. However, if the probe anneals to a specific sequence on the target gene, it will be degraded by the 5’-3’ exonuclease activity of Taq DNA polymerase. The fluorescent reporter will be then released from the quencher which will be no longer close enough to absorb the emission from the reporter dye. The fluorescent signal is thus generated and detected by CCD camera in a LightCycler.

The threshold cycle or. Ct value is the number of cycles required for the fluorescent signal to cross the fluorescence threshold (the real signal could so be distinguished from background signal). Ct level is inversely proportional to the amount of target RNA in the sample. The lower is the Ct level the bigger is the amount of target RNA in the sample.

Abbreviations
qRT-PCR: quantitative Reverse Transcription-Polymerase Chain Reaction
cDNA: complementary DNA
F: fluorophore
Q: quencher
Ct: threshold cycle

References
1- Filion, M., 2012. Quantitative Real-Time PCR In Applied Microbiology. Norfolk: Caister Academic Press.
2- Corman VM, Landt O, Kaiser M. et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25(3):pii=2000045.
3- World Health Organization.


 Diagnosis-of-SARS-cov-2



 

                                     

 

 

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