PCR testing is a type of test that people use for diagnosis. They have been using it for a long time in different crises. These tests have been in use since the early days of the outbreak of the coronavirus pandemic. However, as the name suggests, rapid tests have only recently been introduced and are quicker and more straightforward. Both are to test whether or not a person has Covid-19.
When performing a PCR diagnostic test, a fragment of the genetic material is detected. The tests are focus on finding a pathogen or microorganism. PCR testing, the use of which is routine wherever medical research or development may take place. The test is based on the heat stability characteristics of a polymerase enzyme.
After testing the sample in laboratories (of suspected people), the result will be positive if RNA is detected. Thus, you will know if the patient has Covid-19. On the other hand, if the PCR technique fails to detect the virus’s genetic material, the person would not be infected.
It is important to note that when there is significant clinical suspicion, another test should be performed. Precautions and procedures ensure that patients don’t have the virus.
When Is PCR Used?
PCR is helpful because you can indicate to the polymerase enzyme which fragment of DNA you want to copy. To use it, you must first know the structure of the genetic material you are interested in. Among other things, people use it to:
- Detect infections caused by pathogenic microorganisms, such as the Sars-CoV-2 coronavirus or influenza in humans, and also Leishmania in dogs;
- To know how much of a microorganism (bacterium, virus, fungus) is in a given food;
- Detect whether a bacterium has specific genes for resistance to a particular antibiotic;
- Measure the expression levels of a gene. Also, understand why bacteria with the same genetic code respond differently. Depending on the conditions, of course.
People also use it in forensic medicine to identify victims or aggressors. Especially in the case of a researcher, he uses this method to identify biological remains.
What Are The Most Common Errors That Can Arise?
Although valuable, PCR is not infallible. When discussing how reliable PCR testing is, sensitivity (probability that a positive sample will generate a positive result) and specificity (probability that a negative sample will generate a negative result) should be considered.
In the case of coronavirus PCR, its sensitivity is about 70%-90%. Its specificity is above 99.5%. The information indicates that there are practically no false positives (negative samples hardly return positive results). But there may be some false negatives (positive sample giving rise to a negative result).
In both cases, the error usually results from a failure either during the sample processing itself. It can also happen due to the performance of the test.
Such errors occur because of a variety of reasons. The most common of them is human error. It is also imperative to know the critical points of the whole process since a failure in any of them could lead to an erroneous result.
Some Critical Points To Take Into Account
Choice of the DNA Fragment
It is essential to define the same fragment to cop. So, suppose you want to detect a specific microorganism in a sample (the SARS-CoV-2 coronavirus in a person or the bacterium Listeria monocytogenes in a food). You must “tell” the polymerase what you are looking for and how to find it.
To do this, you must design small fragments of DNA -called primers- that delimit our fragment and tell the polymerase where to start and finish copying. If you want to detect the coronavirus, you will have to copy an utterly different fragment from the one you would need if you wanted to detect L. monocytogenes. Therefore, specific primers will have to be designed in each case.
Suppose we continue with the example of coronavirus detection. It is crucial to collect the sample in the right location and at the right time. In the case of coronavirus, a Doctor needs to take nasopharyngeal samples. In the oral cavity, samples are more challenging to be found.
But the time at which people collect the sample also plays a role. There are situations when a negative result is possible. Days later, the patient may end up presenting symptoms.
This step consists of collecting the genetic material, i.e., removing everything that does not interest us from the sample. This is the case with the remains of the swab from the sample taken when testing for coronavirus. It is also possible with the remains of the processed food, in the case of L. monocytogenes.
The doctor places the sample in a liquid diluent that facilitates the whole process and is heat-treated to break up the cells. Afterward, several other processes need to be followed.
In addition, the starting genetic material may be RNA. In such cases, a step before amplification would have to be carried out, converting the RNA into DNA.
Disclosure of the Results
Finally, to know if the copies are correct, you need to reveal the results. For this purpose, you can have two basic types of PCR:
- Conventional PCR: With a conventional PCR technique known as agarose gel electrophoresis is used to collect samples. This tool allows us to separate the copied fragments according to their size.
If the sample is positive for a microorganism or has a gene you are looking for (e.g., resistance to a particular antibiotic), a line will appear on the gel corresponding to our fragment of interest. When no line is visible, the result would be negative.
- Real-time PCR: in this case, the process is slightly different since you should add a component at the beginning of the reaction. The compound used is fluorochrome. The compound emits a fluorescence each time a new copy of the fragment is made.
A machine capable of measuring these fluorescence levels throughout the amplification process. Samples will be favorable if they exceed a preset fluorescence limit.
Related Topic: Predictive COVID-19 Data Tools
This technique also makes it possible to determine the concentration of a given microorganism in each sample, depending on how many amplification cycles it takes to exceed this limit. The higher the microorganism concentration in the sample, the lower the number of cycles required for fluorescence detection.