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RT PCR Machines in the diagnosis of COVID-19
What are RT PCR Machines and a PCR test??
The RT PCR Machines allow for identifying specific amplified DNA samples by using the analysis of their melting temperature. PCR is a test that is usually carried out to detect the presence of genetic material from a virus. The PCR test has been designed to detect the presence of a virus during a test. The tests also detect the presence of fragments of the virus even after the individual has healed after the infection.
Challenges of using the RT PCR Machines in Diagnosis of Covid 19
According to the National Library of Medicine, there is some controversy surrounding the real-time PCR tests, as there are risks of the tests eliciting false-negative and false-positive results. According to reports a couple of the suspected cases that exhibited typical clinical characteristics of Covid 19 and the identical specific computed tomography were not diagnosed.
Meaning that a negative result does not exclude the possibility of Covid 19 infection, which means that it should not be used as the sole criterion for treatment or for coming up with patient management decisions. There were, therefore, a few factors that were proposed and which should be associated with the inconsistency of real-time PCR.
It is common knowledge that the results derived from the real-time PCR using primers in different genes can be compromised by the variation of the viral RNA sequences. The false-negative results might therefore occur through mutations in the primer and the probe-target regions in the SARS-CoV-2 genome.
And even though it was attempted to create the real-time RT-PCR assay as precisely as possible but largely based on the conserved regions of the viral genomes. The variability that causes mismatches between the primers and probes, and the target sequences could potentially lead to a decrease in assay performance and potential false-negative results.
So, in light of the above, multiple target gene amplification is recommended to minimize the chances of invalid results. Most importantly is that there are several types of the SARS-CoV-2 real-time RT-PCR kit that have been developed and approved rapidly but all of which have different qualities. Notable, therefore, is that the sensitivity and the specificity of the real-time RT-PCR test is not 100%.
In reference to the natural history of Covid 19 and the viral load kinetics observed in different anatomic sites of the patients. The procedure commonly used for sampling has been established to contribute to false and negative results. In a study, it was reported that sputum was the most accurate sample for laboratory diagnosis of Covid-19 and which was followed by the nasal swabs. Notable is that the throat swabs were not approved for the diagnosis.
What’s more, is that it was suggested that the detection of viral RNAs in bronchoalveolar lavage fluid if the specialist needed to diagnose and monitor viruses in very severe cases. In other studies, it was indicated that the only way to obliterate inconsistent results was to use different specimen types such as stool and blood. Also, the extracted samples should be retrieved using the Dacron or polyester flocked swabs and be taken to the lab immediately.
What are The Differences between Rapid PCR vs. RT-PCR Covid Testing?
The PCR test has been designed to detect the availability of a virus’s genetic material or its fragments as it breaks down. Antigen test, on the other hand, is designed to detect particles of proteins on the surface of the virus referred to as antigens. Antigen tests are faster and would normally take between 15 and 30 minutes.
Individuals can also perform the rapid antigen tests which have been established to be more accurate only when used within a few days of the start of the Covid 19 symptoms. And which is normally the time when the body has the largest amount of viruses. The information consolidated above brings us to our next question, between the PCR and antigen test which one is more accurate?
Well, the antigen test has been established to be faster but it is definitely not as sensitive as the PCR test. Given the above, if an antigen test is negative then only a PCR test can be done to confirm the negative PCR test.
The difference between the Rapid PCR test and the RT-PCR Covid Testing is that the former has been designed to detect a new Covid infection through a nasal swab. And will efficiently take note of genetic material belonging to the Sars-Cov-2 virus. The Rapid PCR test produces results on the same day and can be carried out within the facility.
The RT PCR test on the other hand also goes by the name molecular test and is administered via a nasal swab and can also be administered through a throat or saliva swab. When compared to the Rapid PCR test, the RT PCR Covid testing is highly accurate and considered the stellar standard of Covid tests. The results from this test take longer and will not be available up until three business days have elapsed.
And once the samples have been extracted either through nasal, saliva, or, throat swab they are sent to the lab for processing.
How do RT PCR Machines work?
An RT PCR machine works by collecting data, as the PCR process takes place. The system has been designed in such a way that it combines amplification and detection in a single step. The above is made possible by utilizing different fluorescent chemistries that have been established to correlate PCR product concentration to fluorescent intensity.
The process of the real-time PCR to combine PCR amplification and detection in a single step gets rid of the need to detect viruses using gel electrophoresis. What’s more, is that it ensures that the method is truly quantitative. When using the real-time PCR systems the fluorescent dyes are normally used to label PCR viruses when thermal cycling is initiated.
The unit utilizes the fluorescence-detecting thermocyclers to amplify specific nucleic acid order and measures their concentration simultaneously.
How is the COVID-19 Virus Detected using RT PCR Machines?
After the performance of the nasal or throat swab, a sample of the Covid 19 virus is extracted, it is then treated with various chemical solutions that help remove proteins and fats, and the process extracts only the RNA present in the sample. RNA is usually a mix of an individual’s own genetic material and the virus’s RNA but only if present.
And with a specific type of enzyme, RNA is reverse transcribed to DNA. The specialists will then add more short fragments of DNA that have been established to be complementary to specific parts of the transcribed viral DNA. During this process, the specialist will check for the presence of the virus in a sample, which is established when the fragments are detected on particular sections of the viral DNA.
Important to note is that a couple of the extra genetic fragments are normally used for building the DNA strands and that is during the amplification stage. Other genetic fragments are normally used for creating the DNA, and to add marker labels to the strand and which are later used to detect the virus.
The above mixture will then be placed in the RT PCR machine and the machine will then cycle through temperatures that heat and cool the mixture and which will trigger a specific chemical reaction necessary for creating new identical copies of the target sections of the viral DNA.
The above is not the end as the cycle is repeated over and over a feature that enables it to continue copying the target sections of the Viral DNA. Important to note is that each of the cycles conveniently doubles the previous number. Meaning that if there were two copies, then they would become four and the four copies would then become eight.
What you must, therefore, understand is that a standard Real-Time PCR setup will achieve up to 35 cycles. Meaning that by the time the process comes to a conclusion up to 35 billion new copies of the sections of viral DNA will have been created from each strand of the virus that is present in the sample. Notable is that while the new copies of the viral DNA parts are created, the labels attach to the DNA strands and then proceed to issue a fluorescent dye.
The fluorescent dye is usually measured by the machine’s computer and provided in real-time. The computer will then track the amount of fluorescence in the sample once each cycle has been completed. Most importantly is that when a certain level of fluorescence is exceeded it means that the virus is present.
Specialists have also been known to monitor the number of cycles that an individual will take to reach the relevant level. The above enables them to estimate the severity of the infection. If the cycles are therefore fewer, the more severe the viral infection is.
What is the advantage of a Real-Time PCR System?
A Real-Time PCR System has been established to be highly sensitive and specific and can, therefore, deliver a reliable diagnosis in as little as three hours. You will, however, notice that most laboratories might take up between six and eight hours.
And when compared to other virus isolation methods the PCR system works exceptionally faster and has a minimized potential for contamination or errors. Given that the whole process is usually carried out inside a closed tube. A real-time PCR system offers an accurate method of detecting the Covid 19 virus.
And given that the Covid 19 virus only stays in the body for a specific amount of time, the real-time PCR system cannot be relied upon to detect the previous infections. A procedure that could help medics understand the development and spread of the virus.
The advantage of using the Real-Time PCR systems is that they are closed tube systems thus do not require any post-PCR processing.
Viruses and Genetic Materials
From our discussion above we have explored the modalities of the RT PCR machines used in the diagnosis of Covid 19. We will now dig into the makeup of a virus so that you understand the differences between DNA and RNA. For starters, a virus is a microscopic package of genetic material effectively covered by a molecular envelope. The genetic material is usually a ribonucleic acid also referred to as RNA or a Deoxyribonucleic acid also referred to as DNA.
DNA can be described as a two-strand molecule available in all organisms including plants, animals, and viruses. DNA holds the genetic code, for how these organisms are made and how they develop. RNA, on the other hand, is a one-strand molecule that replicates various parts of the genetic code to proteins to synthesize and carry out functions that keep organisms alive and developing. Important to note is that there are different variations of RNA that copy, transcribe and transmit.
So some of the viruses the likes of the SARS CoV 2 coronavirus responsible for causing Covid 19 have been determined to only contain RNA. The above then means that the Covid 9 virus relies on infiltrating the healthy cells so that they can survive. Private medical practices or clinic Surviving During the Coronavirus Pandemic.
The moment the virus manages to penetrate the body cells it uses its own genetic code, “ the RNA” to take over the cells, it will then control and reprogram the cells consequently turning them into virus-making factories.
Now here is where the RT PCR machines come in, for the Covid 19 virus to be discovered early in the body using real-time RT-PCR, the specialists have to convert RNA to DNA. The above process is known as Reverse Transcription and is done because only DNA can be replicated.
The next big question is why scientists amplify DNA.
A specific part of the transcribed viral DNA has amplified hundreds of thousands of times, which provides them with a large enough quantity of the target sections of the viral DNA which aids in confirming that the virus is actually present.
Which are the best RT PCR Machines?
The best PCR system is the Rotor-Gene Q-Pure detection real-time PCR system because it is future-proof and designed to offer outstanding performance. The units come complete with a wide optical range and flexible formats.