PCR Detection of COVID-19 (SARS-CoV-2)

PCR Detection of COVID-19 (SARS-CoV-2)

Contents

Background

Assay Information

Sample Types

      Upper Respiratory

      Lower Respiratory

Notes

Background

COVID-19 (causative agent: SARS-CoV-2), the novel coronavirus which has brought the capital of China’s Hubei province to a standstill, has been deemed a Public Health Emergency of International Concern by the World Health Organisation as of January 2020. (1) Wuhan is the epicentre of the epidemic, but cases have been reported across the world. A large portion of cases outside China have been contracted by people who were travelling in mainland China, although there have been reports of locally-transmitted disease acquisition (Ref).

According to the 23rd of February WHO situation report, there have been a total of 78,811 confirmed cases and almost 2,500 deaths. (Ref)

The gravity and death toll of COVID-19 begets significant need for quick and accurate detection of the novel Coronavirus strain (Ref). SARS-CoV-2 is closely related to other viruses in the SARS-CoV clade, and thus tests need to be sensitive and specific enough to correctly determine the strain of the virus.

Virus & Disease Name

There has been some confusion regarding the naming of the disease and of its causative viral agent. The World Health Organisation (Ref) has recently released the following information, represented visually in the table below:

Name of Viral Agent Name of Disease

Official

Severe acute respiratory syndrome coronavirus 2

Coronavirus disease

Abbreviation

SARS-CoV-2

COVID-19

In public communications:

“The virus responsible for COVID-19”

“The COVID-19 virus”

COVID-19

Fig 1. World Health Organization COVID-19 related names.

It is important to note that public communications tend to refer to the viral agent without using the official name it was given by the International Committee on Taxonomy of Viruses. This is because the virus name (SARS-CoV-2) was thought to “have unintended consequences in terms of creating unnecessary fear for some populations, especially in Asia which was worst affected by the SARS outbreak in 2003.” (WHO)

Assay Information

The Assay Genie Coronavirus COVID-19 (SARS-CoV-2/ Strain 2019-nCoV) Triplex RT-qPCR Detection Kit  can be used for the in vitro detection of COVID-19 in respiratory specimens (sputum; nasopharyngeal, oropharyngeal aspirates, washes or swabs; tracheal aspirates). The kit uses real time (RT) qPCR.

The comprehensive kit allows efficient cDNA synthesis and Real-Time PCR in a single tube. The kit includes a qPCR MasterMix supplied in a 2x concentration to perform real-time PCR. qPCR MasterMix contains all the reagents needed for running PCR reactions (barring PCR primers, probe and template). In addition, a separate RT Mix comprised of a balanced mixture of Reverse Transcriptase and RNase Inhibitor is also provided.

The kit contains one set of primers and fluorescent probes to differentiate between 2019-nCoV/SARS-CoV-2 and SARS-CoV (or bat SARS related CoV). Coronavirus 2019-nCoV RNA targets are amplified and detected in the FAM channel, while Coronavirus SARS-CoV RNA targets are amplified and detected in the HEX, VIC or JOE channel (depending on the equipment used).

The kit also includes a positive and negative Control. The positive control is supplied to demonstrate that the PCR amplification is working efficiently with the supplied components. To confirm absence of contamination, a negative control reaction should be included every time the kit is used.

Sample Types

Upper Respiratory

  • Nasopharyngeal Swab
  • Nasopharyngeal Wash & Aspirates
  • Oropharyngeal Aspirates

Lower Respiratory

  • Sputum
  • Tracheal Aspirates
The nasopharynx and oropharynx are two common targets for upper respiratory samples.  The picture shows their relative placement in the upper respiratory tract.

Fig 2. The nasopharynx and oropharynx are two common targets for upper respiratory samples. (NIH National Cancer Institute)

Nasopharyngeal Swabs

Nasopharyngeal swabs and aspirates represent some of the most ubiquitous tests for the detection of respiratory viruses. A swab test typically involves the use of a sterile dacron or rayon swab with a plastic shaft to collect a sample, while an aspirate or wash uses a syringe with a flexible catheter to extract a sample.

A full written protocol can be found here for nasopharyngeal swabs, and The New England Journal of Medicine video below shows an example of the procedure.

 

For both nasopharyngeal swab and aspirate specimen collections, the U.S. Center for Disease Control (CDC) page here gives an overview of the technique and procedure.

Nasopharyngeal Wash & Aspirates

For children, a nasopharyngeal wash may be preferable to a swab. This procedure entails a sterile saline wash to aid in the collection of epithelial cells from the nasopharynx. (Ref)

Image shows a patient undergoing a nasopharyngeal wash, in this labeled diagram.

Fig 3. A patient undergoing a nasopharyngeal wash. (St. Jude’s Children's Research Hospital)

Oropharyngeal aspirates

To collect an oropharyngeal swab, a sterile swab is inserted into the posterior pharynx and tonsillar areas via the mouth. Then, the swab is rubbed over the tonsillar pillars and posterior oropharynx.

It is important to avoid touching the tongue, teeth, and gums during the specimen collection. The Pan American Health Organisation (a branch of WHO) has a video detailing the procedure here.

The full CDC guideline for specimen collection can be found here.

Sputum

Sputum is mucus which has been produced by and subsequently expelled from the respiratory system by coughing. Functionally, it is phlegm that has reached the outside world. (Ref) Expectoration is a verb which encompasses coughing or spitting, and sputum production is the act of expectorating (coughing and spitting out) the substances produced within the respiratory tract. (Ref)

Generally, these specimen collections involve rinsing the mouth, then having the patient cough deeply into a sterile collection container. It is important to note that the sample should not contain any mucosal contaminants from the nose, and ideally very little residual oral mucous.

Tracheal Aspirates

Sputum is the preferred method of gathering specimens from the lower respiratory tract for testing. In such cases where there is medical precedent or emergency, tracheal aspirates can be collected if a tracheostomy has taken place. As a result of the invasive nature of the procedure, alternatives such as sputum are generally preferred. (Ref)

Notes

This kit is intended for research use only. Samples should be stored in accordance with regional guidelines, and such specimens should only be taken by trained professionals.


COVID-19 detection assay for the rapid detection of COVID-19 in patient samples.
COVID-19 (SARS-CoV-2) Triplex RT-qPCR Detection Kit (VA0001)
23rd Apr 2020 Paige Dougherty, MSc. Immunotherapeutics

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