Nucleic acid testing for COVID-19

This is a replay of our biotechnology training workshop which was held on 08/27/20.

You may have heard a lot about RT-PCR testing for COVID-19 in the news. But how does the test work, and what do the results mean? In this livestream, we discuss this technique and demonstrate a BRAND NEW electrophoresis experiment. We'll study the molecular biology behind this technique, including an in-depth look at the polymerase chain reaction. Finally, we'll discuss why COVID-19 testing is important for public health.

For the slides: https://www.edvotek.com/site/pptx/edv...
For the RT-PCR simulation kit: https://www.edvotek.com/123
For the immunoassay simulation kit: https://www.edvotek.com/1219

Have a question? Email us at [email protected].

Viruses are simple infections particles that cannot replicate independently – they are dependent on the cellular machinery within their specific host. Because viruses carry genetic material, reproduce and evolve but rely entirely on a host organism for their basic biological functions, they’re considered to be on the border of biology and chemistry.

The term ‘Coronavirus’ actually refers to an entire family of viruses. Coronaviruses like SARS-CoV-2 have a single-stranded RNA genome wrapped in a helical capsid. A membrane envelope surrounds the capsid. The capsid and the membrane protect the RNA genome from the outside environment.

The envelope is studded with proteins that help the virus infect cells. They interact with receptors on the surface of the cells in our respiratory system. This lets them invade the cells, where they take over the cell’s machinery and reproduce.

In today’s medical diagnostics laboratory, researcher use a technique called the polymerase chain reaction, or PCR to identify the presence of the viral genome. This is an incredibly powerful technique that can take the smallest amounts of DNA and expands it to be used for analysis.

But, there is a problem. Taq polymerase is a DNA-dependent DNA polymerase, meaning that it builds new DNA using old DNA as template. It cannot use the RNA genome of SARS-CoV-2 as a template because there are differences in the actual chemical structure of the bases and the backbone that connect them.

In order to detect COVID-19 using PCR, Reverse Transcriptase (RT) is used to synthesize complementary DNA (cDNA) copies of the RNA genome. A small amount of the cDNA is mixed with Taq polymerase, dNTPs and primers for amplification by PCR, and then the samples are analyzed.

Most molecular tests target several locations within the SARS-CoV-2 genome. In our test, we are targeting two regions in the SARS-CoV-2 N protein gene. As an internal control, we amplify the human gene RNase P which is a housekeeping gene, or a gene that is always turned on because it is responsible for normal, day to day activities in the cells. But you may be asking, isn’t this a viral test? Well, yes, but remember our sample came from the top of our throat, and was a mixture of human and viral material. So, human DNA will be extracted, copied into our cDNA, and can be amplified by PCR.

All three of these primer sets are added to one PCR sample. The test produces different results depending upon whether the virus is present in a patient sample. We’ll see one band – the housekeeping gene – if a patient is negative. There will be three bands – two from the virus and one from the human – if the patient is positive. If the internal control is missing, the results are inconclusive and the test must be repeated. These results analyzed using fluorescent probes found in quantitative PCR, or electrophoresis.

Because RT-PCR is extremely sensitive and can detect very low levels of the virus, it is considered the “gold standard” for SARS-CoV-2 detection. However, since RT-PCR tests are performed in a medical diagnostic laboratory, it may take several days to get the results, even though the actual test takes a few hours.