In the course of the last year a multitude of COVID tests have been developed or are in the process of being developed. But, it is the PCR test, for the detection of SARS-CoV-2, that has been the central talking point.
The classic PCR test was developed in the 1980s and was a major milestone in laboratory diagnostics. It is a standard procedure for the detection of viruses that is based on the polymerase chain reaction (PCR), in which the genetic material of the pathogen is amplified. Viruses can be detected with this method, even if there are only a few present in the body. PCR tests have a high sensitivity because they can detect the virus or its components accurately. And since only the DANN of SARS-CoV-2 is amplified, the test also has a high specificity. Meaning, it only detects this desired pathogen.
The originally developed PCR test was complex due to the many steps it took to get a result. Especially, the repeated cooling and heating phases were difficult to manage. An important step in the development of PCR was the introduction of thermostable DNA polymerases, which simplified and accelerated the method. As time went on, more specific and refined analyses has become possible.
Real-Time RT-PCR-Test
The real-time, gold standard, RT-PCR test is especially well suited to determine the quantity of the amplified DNA section. For example, it is used for the detection of HIV, influenza and SARS-CoV-2 viruses. The term RT-PCR is reserved for the reverse transcription PCR test, an entirely different method. More technically, it is also called the qPCR test.
The special thing about the qPCR method is that it responds visually;
A fluorescent dye is added to the actual reaction and through DANN amplification, the dye becomes visible. The fluorescence is determined in real time, each time the viral DNA replicates, hence the name ‘real-time’. The number of replications can be determined from the fluorescence and
statistical methods can also be used to derive the number of copies per reaction and quantification is possible. For example, the correlation of the virus copies with the infection level can be calculated. The continuous improvement of test sensitivity and specificity remains a research goal, especially during a pandemic. The qPCR is the most widely used COVID test, but there are also other ways to detect SARS-CoV-2.
LAMP-Test (Loop-mediated Isothermal Amplification)
An important difference between LAMP and qPCR is the amplification of DNA segments at a constant temperature; isothermal amplification. This is made possible because the DNA is applied in a strand-shifted manner with no need for high heating to separate the DNA strands. This eliminates the time consuming heating and cooling phases that are necessary for qPCR tests making the results more readily and quickly available. A benefit of the LAMP test is seen in quicker amplification of virus DNA; Other enzymes are used compared to the qPCR test. A disadvantage of the LAMP test is its reduced accuracy compared to the qPCR test.
Further studies have spoken favourably of LAMP tests due to the faster speed and lower cost. As of 16 March 2021, detailed studies on the use of LAMP tests for SARS-CoV-2 are not yet available. With the exception of the thermocycler for temperature control, the LAMP test requires the same chemical testing equipment as conventional PCR tests, of which has caused controversy in German laboratories; nucleotides and plates are only offered by a few companies, there is competition between the LAMP and PCR methods. Additionally, most medical laboratories are set up on a large scale for PCR testing and switching to LAMP would require renewing or adapting the entire laboratory process, including equipment and software. Additionally, staff would need retraining and the guidelines for accreditation would need to be guaranteed.
This is why LAMP is not yet well established in German medical laboratories; The RT-LAMP test is mainly used in the United Kingdom to test for SARS-CoV-2.
Multiplex tests for the identification of multiple pathogens
Multiplex tests can test for several pathogens at the same time. In this method, influenza can be distinguished from an illness that is caused by SARS-CoV-2. These tests can test for between two and twenty different pathogens simultaneously. Depending on the complexity, the multiplex test in particularly interesting for co-infections and it can also be cost-intensive. The benefits and costs of this test must be weighed up carefully. For example, multiplex tests are relevant for intensive care units, as the pathogens and possible co-infections can be identified by using only one test, resulting in a quicker reaction time.
SARS-CoV-2 diagnosis with nanoparticles
Another approach to testing is nanotechnology-based. This method is based on magnetic nanoparticles coated with viral antigens. To perform the test, blood is applied to the test surface and the nanoparticles are then treated with a developer reagent. If the sample contains antibodies against the coronavirus, a colour change occurs. While the traditional ELISA test, antigen test, takes about three hours to show any results, studies have shown that this new method only takes a few minutes.
Therefore, nanotechnology-based diagnostics offer an alternative for quick and easy detection of antigens.
Rapid corona tests for home use
Antigen tests provide results in a few minutes. They are inexpensive to produce and do not need to be evaluated by a laboratory. They are particularly suitable to detect an infection early on, when the viral load is particularly high, and the infection rate is also high. However, later on in the infection they are less reliable. As a comparison, a PCR test can detect a single RNA molecule in a microlitre of solution. An antigen tests needs thousands of viral proteins per saliva sample to identify a positive sample. The test may give a false result if there is only a small number of SARS-CoV-2 viruses present in the body or in the sample.
The outlook of rapid testing
The current pandemic has shown the importance of efficient diagnostics. qPCR is the most widely used method to test for SARS-CoV-2 and due to its high sensitivity, the test provides a reliable result at any stage of a COVID infection. However, a test result takes 4 to 6 hours and requires a laboratory diagnosis. Equipping German medical laboratories with LAMP technology could increase test capacities due to their faster results. Further research and application in the area of nanotechnologies could produce rapid and more cost-effective solutions in the field of rapid testing. Zamann Pharma Support GmbH works with renowned development companies and manufacturers in Europe, and specializes, among other things, in the distribution of PCR tests and associated equipment. From hygiene concepts to constructing test centers, Zamann is your professional partner to actively support you every step of the way.
Sources
fda.gov | bundesgesundheitsministerium.de | pagepress.org | nature.com
