By Linya Thng
With death tolls rising higher than ever, researchers are urged to seek alternative methods for coronavirus diagnosis in hopes of returning to normality. Currently, in status quo, viral diagnostics are heavily relying on nasal-swab samples. However, whilst having an almost 100% accuracy in identifying infected people has, this method has its drawbacks. These high sensitivity PCR tests require trained personnel, specific reagents, and expensive machines that take hours to produce results. In other words, not an ideal picture for many developing countries. The same goes for developed countries, for example, the United States leads the world in COVID-19 death but severely lacks in testing capacity. Several countries are struggling to ramp up testing to accommodate the second wave of COVID-19. Is there a faster, simpler, and cheaper alternative?
Currently available tests for COVID-19 can be categorized into 1) diagnostic tests (such as antigen and PCR assays) which detects SARS-CoV-2 virus and 2) antibody tests that detect molecules produced by infected people. However, antibodies require several days to develop after infection and remain in the blood for weeks after recovery, hence limiting the effectiveness of antibody tests in diagnosis (Guglielmi, 2020). The probability of a positive result varies with each test before and after symptoms appear.
A majority of the tests utilise nasal-swab samples where viral RNA is amplified to a detectable level and its presence flagged. This method revolves around detecting, amplifying, and labelling the viral RNA and uses pieces of sequences (called primers), DNA-building enzymes, and a stock of DNA ‘letters’. This method is also known as reverse-transcriptase polymerase chain reaction (RT-PCR). In summary, a computer measures the fluorescent signal, which flags the presence of the virus and hence concludes in a positive test result (Guglielmi, 2020). The gold-standard diagnostic test uses RT-PCR and works by identifying a specific SARS-CoV-2 genetic sequence from a sample taken from cells or fluid in a person’s nose/throat. If the viral sequence is detected, the technique amplifies it to levels that enable it to be detected. Firstly, the virus RNA is converted to DNA. Primers (short designed DNA sequences) perform several functions – some tag specific sections of the viral genetic code that assists in the duplication of the sequence which involves repeated heating and cooling. This process of amplification allows the detection of the virus to be a lot more accurate and efficient. Other primers can label amplified DNA strands. These labels release a fluorescent signal which is detected by a computer, flagging the presence of the virus.
Various approaches aim to reduce the time taken to provide a test result, for example, by amplifying the DNA at a constant temperature, which eliminates the necessity for repetitive rounds of cooling and heating. Some are existing assays customized to identify and detect SARS-CoV-2. Healthcare companies in the US have developed coronavirus assays that run on smaller hardware platforms and takes up less time to perform. However, reagents and platforms come at a price, and slight tweakings can deter the devices from accurately detecting the virus.
Numerous tests are based on a technique known as loop-mediated isothermal amplification (LAMP) which works at a constant temperature and has previously been used to identify viruses such as Zika (Yu et al., 2020). LAMP relies on two enzymes – one to copy DNA and one to convert viral RNA to DNA. It also requires a set of 4-6 short primers designed to recognize different snippets of the viral genome. These fragmented initiates copying and also allow newly copied DNA strands to form looped structures that can be amplified at a rapid rate compared to standard PCR. This method amplifies the viral sequence by coaxing it into loops of various shapes. The drawbacks of this method are that only a few dozen samples can run at a time and is less accurate.
A different approach for faster and cheaper diagnostic tests would be to take a different approach: rather than attempting to detect the viral genome, we could look for molecules that sit on the surface of the virus. Such a test would involve an antibody specifically tailored to a certain protein or antigen (similar applications to pregnancy tests). These assays are also inexpensive to produce, easy to use and have already been used to detect influenza infections. Another obstacle would be scaling up the assays for mass production. With that limitation, a small number of tests can hopefully be deployed before the end of the year. Alongside with the gold standard, there is a potential country that can reach its target of conducting millions of tests per week and prepare for any future scenarios.
References:
Guglielmi, G. (2020) The explosion of new coronavirus tests that could help to end the pandemic. Nature (London). 583 (7817), 506-509. Available from: doi: 10.1038/d41586-020-02140-8.
Yu, L., Wu, S., Hao, X., Dong, X., Mao, L., Pelechano, V., Chen, W. H. & Yin, X. (2020) Rapid Detection of COVID-19 Coronavirus Using a Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) Diagnostic Platform. Clinical Chemistry (Baltimore, Md.). 66 (7), 975.
Imperial Bioscience Review 