The emergence of the new coronavirus pandemic has created an unprecedented threat to human lives worldwide. The scale and severity at which this epidemic is growing is beyond the imagination of researchers around the world. As the biopharmaceutical and conventional pharmaceutical industries scramble to find a cure or a vaccine for this disease, there is an urgent need for rapid detection technologies that can enable quick and cheaper detection methods with utmost fidelity.
Currently two core technologies — polymerase chain reaction (PCR) and antibody testing — form the mainstay of the global testing system against the COVID-19 virus. Both these technologies have their own advantages and disadvantages.
The real-time reverse transcription polymerase chain reaction (RT-PCR) detection test finds the presence of nucleic acid from SARS- CoV-2 in samples like nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract aspirates, and nasopharyngeal wash/aspirate or nasal aspirate.
COVID-19 antibodies are produced by the host immune system following exposure to the new coronavirus. IgM is the first antibody that appears at the onset and IgG is involved in the secondary immune response. The test involves an ELISA microplate which is coated with nucleocapsid (N) protein (a recombinant protein expressed in E coli) and purified. The coated N protein mixing binds with IgG/IgM N antibodies in the serum sample. In this case, a secondary antibody-HRP conjugate is added, which binds to pre-formed complexes of IgG/IgM antibody-N antigens. On the addition of the substrate, a coloured reaction takes place.
This COVID-19 IgG/IgM ELISA assay is qualitative. The sample of choice, in this case, is blood.
The third type of technology which is an extension of the antibody-based assays is the lateral flow assay, which is used as a tool for carrying out serological epidemiological investigations.
Despite these tests, there is an urgent need for the research world to find alternate ways for rapid, point-of-care detection.
The ELISA-based serological test for antibodies and the PCR-based test are not very feasible as far as mass testing and/or point-of-care testing is concerned. In contrast, lateral flow methods need only 10-15 minutes and results can be read visually without the need of an analyser. The principle behind this assay is based on the use of a test cassette. This cassette contains recombinant antigen of the COVID-19 virus which is conjugated to coloured particles. During the test, when the sample is added to the cassette`s sample well, IgG and IgM present in the sample binds to the antigen, forming a coloured coronavirus antigen-antibody complex. This complex then migrates laterally over the strip towards the other end. In the test region of the strip, anti-human IgM and anti-human IgG are already present. This causes lines to appear if the sample is positive.
Methods of choice
The technology used in RT-PCR-based kits is well established. RT-PCR tests are used to directly detect the presence of an antigen/RNA, rather than antibodies. By detecting viral RNA, which will be present in the body before antibodies form and symptoms manifest; the tests can tell whether or not someone has the virus very early on.
As mentioned earlier, RT-PCR involves 3 levels of testing which focus on finding multiple target sequences simultaneously and therefore it will remain the backbone of testing facilities. Therefore, RT-PCR based kits are the most suited for efficient and sure-shot testing as it involves testing for multiple genes with positive and negative controls and can also give an indication about the stage of the infection.
Another possibility is the use of novel RNA sequencing technologies like Oxford nanopore, but currently, the per-sample cost for this method is too high. Another approach is the newly introduced molecular diagnostic approach. Here, RNA detection is done using loop-mediated isothermal amplification (LAMP), followed by simple visual detection of amplification. This test is rapid and reliable, and involves the amplification of a small amount of target sequence at a single reaction temperature which avoids the need for sophisticated thermal cycling equipment. Both these RNA testing methods can pay rich dividends if pursued extensively.
Unique technologies in the making
The RT-PCR based detection system involves three main steps, namely, sample collection (saliva, sputum, swabs), followed by RNA extraction and finally RT-PCR based sensitive detection. There is still some scope for improvement in these steps, which will greatly enhance its capabilities and reduce the turnaround time.
In this regard, one innovation which will help in improving RT-PCR based testing is the introduction of rapid RNA extraction and a potent viral collection/transfer buffer. MagGenome Technologies Pvt Ltd is working on both these aspects. MagGenome’s unique and patented RNA extraction protocol from oral samples provides quick RNA extraction, yielding 3-4 times the recovery as in half the time compared to other existing technologies. In addition to this, MagGenome has a unique collection buffer in which oral samples can be collected and transferred at room temperature. Both these innovations can greatly improve the robustness of RT-PCR based testing strategies.
Another quick way in which nanoparticles can be utilized to make a point-of-care detection system is by immobilising COVID-19 antigens on the surface of magnetic nanoparticles. This nanoparticle-antigen conjugate can then be added to the sample like blood or serum. If IgG and IgM are present in the samples, it will lead to the formation of nanoparticle-antigen-antibody complex, which can be easily separated using a magnetic stand. This will remove any contaminant that affects the sensitivity of the test. A suitable secondary antibody labelled with an enzyme or with a fluorescent dye can then be added to this conjugate for quick and sensitive detection.
One such promising technology is a magnetic nanoparticles-based technique which is more efficient as well as time and cost-effective. These advantages are attributed to the use of bare/uncoated magnetic nanoparticles. This is also a key differentiating factor compared to other approaches that use either silica columns or coated beads. Magnetic nanoparticles have unique surface properties with high magnetic susceptibility, making them very useful in genomic and proteomics applications as well.
The author is CEO, MagGenome Technologies Pvt Ltd