COVID-19 (SARS-CoV-2) Assay Tests
Detection Methods for SARS-CoV-2
Coronaviruses are enveloped positive-sense RNA viruses that belong to the Orthocoronavirinae family that are distributed broadly among humans, other mammals, and birds and that cause respiratory, enteric, hepatic, and neurologic diseases source. Although there are six known species of this virus that are known to infect humans, however, only four are prevalent and usually cause cold-like symptoms source. These include 229E, OC43, NL63 and HKU1. The other two species of viruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic viruses and have previously caused major pandemic incidents in 2002-2003 and 2012 source.
Figure 1: Schematic labelled diagram of Coronavirus (SARS-CoV) (Taken from Peiris et al., 2003)
In December 2019, a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) was first isolated in Wuhan, China. Genetic analysis conveyed that SARS-CoV-2 is closely related to SARS-CoV, the virus that caused a pandemic in 2002-2003 and is a part of the genus Betacoronavirus source. It is an enveloped positive-sense RNA virus, and contains four major structural proteins which include the spike (S), membrane (M), envelope (E) and nucleocapsid (N) source. A recent study confirmed that angiotensin converting enzyme 2 (ACE 2), a membrane exopeptidase, found mainly in the capillaries of the lungs, is the receptor used by SARS-CoV-2 for entry into the human cells.The virus has been revealed to enter cells via endocytosis and membrane fusion. The virus causes coronavirus disease (COVID-19) and the symptoms associated with the disease include:
- Dry cough
- Shortness of breath
- Death in severe cases
COVID-19 is spread primarily via respiratory droplets and can infect people via their eyes, nose or mouth and this can occur when the droplets land directly on somebody’s face or when those susceptible touch their face with contaminated hands. In addition, coronavirus transmission can also occur via fecal-oral or body fluid routes including sweat or urine source. WHO reported that the coronavirus infection affects lungs in three phases, viral replication, immune hyper-reactivity and pulmonary destruction source. The virus mainly infects the upper airways of the lungs and can infect and divide in mucous producing pulmonary cells or ciliated cells source. The virus can reach a concentration of about 100 million particles per milliliter in phlegm source. and can survive on contaminated surfaces and objects at room temperature for up to six days source.
Detection Methods for SARS-CoV-2
PCR techniques for viral detection and quantification offer the advantages of high reproducibility and sensitivity (Watzinger et al, 2014). PCR is a tool used to measure the quantity of genetic material (DNA or RNA) in a sample. It involves the use of an enzyme, usually Taq polymerase, which amplifies a short specific part of the template DNA in cycles. In each cycle, a number of small specific sections of DNA is doubled which leads to the exponential amplification of targets. The number of cycles in a PCR experiment is usually between 30-45. Reverse-transcriptase PCR (RT-PCR) is used to detect RNA, as the RNA is reverse transcribed to DNA source.
In RT-qPCR, the same method occurs with the exception of two factors. Firstly, the amplified DNA is fluorescently labelled and secondly, the amount of fluorescence released during amplification is directly linked to the amount of amplified DNA source. Therefore, the amount of DNA within a sample can be measured by the amount of fluorescence produced. The Assay Genie Coronavirus COVID-19 (SARS-CoV-2/ Strain 2019-nCoV) One-Step RT-qPCR Detection Kit is a useful method for the in vitro detection of COVID-19 in respiratory specimens using RT-qPCR.
The kit allows efficient cDNA synthesis and Real-Time PCR in a single tube. The kit includes a qPCR master mix supplied in a 2X concentration to perform real-time PCR.
The kit also contains one set of primers and fluorescent probes to differentiate between SARS-CoV-2 and SARS-CoV (or bat SARS related CoV) and a positive control (PTC) and negative control (NTC). 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.
Immunofluorescence ELISAs are reported to inconsistently detect antibodies produced against viruses before day 10 or 20 after the onset of symptoms respectively source. Therefore, although these methods are useful when tracking the course of infection, they are less accurate at detecting infection at early stages. In contrast PCR-based assays such as RT-qPCR are more accurate and have the potential to detect infection at an earlier stage. Earlier detection of infection may provide potential for the implementation of therapeutic measures or interventions, such as quarantine, and these measures may reduce the risk of person-to-person transmission source.
The next method used to detect viruses in a sample are immunofluorescence assays. It has been used extensively to directly detect a variety of viral antigens.Immunofluorescence use antiviral antibodies to detect viral antigens in tissue sections or infected cells. Infected cells such as those from the mucous membrane of the upper respiratory tract or cells that are present in the mucus aspirated from the nasopharynx can be used. Immunofluorescence assays use a fluorescent label that is conjugated to the antiviral antibody, which is known as direct immunofluorescence or to an anti-antibody, known as indirect immunofluorescence. The amount of fluorescence seen can be viewed with UV light. The amount of binding of the antibody to the antigen is directly correlated with the amount of fluorescence produced source.
A chest CT is another detection method for determining the presence of SARS-CoV-2 in a patient who’s lab results have returned as negative despite the patient having the associated symptoms with the virus. Specific features that are associated with the presence of SARS-CoV-2 include ground glass opacities in the lungs which are caused by increased lung opacity through which the vessels and bronchial structures can still be seen. In addition, interstitial thickening or partial collapse of lung alveoli can also be seen. source.
Figure 2: (A):Chest X-Rays showing glass ground opacities highlighted by black arrows. (B): Chest X-ray with no ground glass opacities. (Taken from Taken from Schulze et al., 2011)
ELISA is the final detection method that can be used to detect if a patient has been infected with SARS-CoV-2. ELISA can detect antibodies or antigens in a sample. If a patient has been infected with SARS-CoV-2, there will be a presence of viral antigens which can be sampled and detected. Antibodies specific to the viral antigens are bound to a plastic surface, and the sample is overlaid. If the sample is positive for viral antigens, they will bind to the specific antibodies. After, a second antibody tagged with a marker is added. If the sample contains the viral antigens it will result in a positive reaction and will be detected by the marker changing colour when the appropriate substrate is added. If no viral antigen is present in the sample, no colour change will occur source. The antibodies of SARS-CoV-2 that can be used in an ELISA detection kit include the spike antibody, envelope antibody, membrane antibody or nucleocapsid antibody.