Everything you need to know.
While the world is busy fussing over the pandemic, medical professionals are stuck on introducing techniques to detect and end the fatal virus ‘Covid-19’. The first main task was to develop something that would detect the virus and, gladly, has already been taken care of. Which is why you are able so easily able to search " find Covid testing near me " and find multiple options! Possibly even one from us, MedNow Labs!
Now let's get to it.
Many methods, including PCR and RT-PCR mainly, are used to detect the virus by amplifying nucleic acids. All such methods fall under the name of NAAT (Nucleic Acid Amplification Test).
What is NAAT, and How Does It Work?
A Nucleic Acid Amplification Test (NAAT) is a specific kind of diagnostic test used to detect various infectious diseases. It consists of high-performance tools for rapid and accurate detection. It is a technique for inspecting the genetic material of the subject being tested. It involves identifying the RNA sequences, particularly in order to check the presence of the genetic material of the virus.
NAAT is used to detect several diseases, including the ongoing battle of Covid-19. Therefore, this article will showcase all the information about NAATs with reference to Covid-19. It would be easier to follow up and understand the whole step-by-step detection process via NAAT this way.
NAAT is widely used to detect the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the virus that causes Covid-19. The specimen collected for testing the subject via NAAT can be from any part of the body depending on the disease being detected. In the case of Covid-19, it is the upper or lower respiratory tract. It can also be done with a saliva specimen, but their quality tends to vary highly. Therefore, the Centers for Disease Control and Prevention (CDC) strongly suggests using and testing the upper respiratory specimen. Some of the examples of upper respiratory specimen include anterior nasal, nasal mid-turbinate, and nasopharyngeal.
Due to the excessive spread of diseases (now the Covid-19), the number and the type of NAATs have increased duly. Different methods of NAATs have been in use in various settings. Some are to be done explicitly within laboratories, others in point-of-care (POC) settings. However, some can also be handled at homes or at other non-healthcare locations under self-administration.
The time of the test result also varies from one NAAT to another. Some of them are such quick procedure tests that you get the result within minutes of finishing the test. However, some others may take time ranging from an hour to a whole day, maybe even more. The most important thing is that every NAAT method carries a different level of sensitivity for detecting SARS-CoV-2 genetic material in a specimen. The strongest sensitivity level, as checked and confirmed, is that of laboratory-based NAATs.
Therefore, laboratory-based NAATs are trusted more and considered more authentic. But, if the circumstances do not allow access to laboratory-based NAATs (due to lack of resources and appropriate help), POC NAATs and other self-administered NAATs can also be used validly.
The many different methods of NAATs used to amplify nucleic acids and detect the virus causing Covid-19.
Polymerase Chain Reaction (PCR)
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Nicking Endonuclease Amplification Reaction (NEAR)
Loop-Mediated Isothermal Amplification (LAMP)
Transcription Mediated Amplification (TMA)
Strand Displacement Amplification (SDA)
Helicase-Dependent Amplification (HAD)
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
Polymerase Chain Reaction (PCR)
The PCR test is a molecular test processed to detect genetic material from a specific organism like a virus. It is considered a standard test to diagnose the fatal Covid-19. It involves inspection of the upper respiratory specimen in order to detect RNA (Ribonucleic acid) of SARS-CoV-2. The PCR technique is used to amplify RNA of the specimen into DNA (Deoxyribonucleic acid). The DNA keeps replicating under observation until SARS-CoV-2 is detected, in case it is present.
Step-by-step Process of PCR test
The PCR test includes three basic steps explained below.
Collecting the specimen
Specimen collection is the first and easy step where a health professional uses a swab (a soft piece of material on a stick or rod) to collect respiratory material present in your nose. The swab can be of several different types. It can be a nasal swab that immediately collects a specimen from your nostrils or a nasopharyngeal swab inserted into your nasal cavity to collect the sample. Once the sample is collected, the swab is put and sealed in a tube and moved to the laboratory for further inspection.
Extraction of genetic material
When the sample is received by a healthcare professional at the laboratory, they separate (extracts) the genetic material of the specimen from the other contents of the material.
This final step of the process involves the amplification of genetic material within the test tube. It is done with the help of several special chemicals, enzymes, and the thermal cycler (a PCR machine). After a number of heating and cooling cycles, the test tube shows millions of copies of the genetic material of the SARS-CoV-2 virus. Now, suppose the SARS-CoV-2 is really present in the genetic material. In that case, it gets detected easily with the help of a chemical that produces a fluorescent light on sensing it in the sample. There are certain indicators that prove the signal is a positive test result sign.
The PCR test is considered highly reliable and accurate for detecting active infection of the SARS-CoV-2 virus. Although most of these tests take hours to be done, some of them are faster too.
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
You might have searched ‘RT PCR test near me’ before. But do you know what it really is? Well, this particular technique is a process carried out inside a laboratory. It involves the combination of reverse transcription of RNA into DNA (named cDNA or complementary DNA). Besides the addition of reverse transcription, the same process of PCR (polymerase chain reaction) continues, and the target DNA keeps amplifying. Further, the amplification process is looked over by the technique of real-time PCR that uses fluorescence.
While PCR practices enzymatic amplification of DNA, RT-PCR involves starting the process with RNA from scratch. Both of the methods have a slight difference. That little difference is the RT-PCR technique having an additional step of reverse transcription of RNA to DNA before the amplification step starts.
The Process of RT-PCR
It is again a simple process that starts with the collection of samples from your body parts like the nasopharynx and oropharynx through a kind of swab. The collected sample is then treated with certain chemicals to extract the RNA from it. This RNA consists of your genetic material as well as the virus's RNA (if present). Here, reverse transcriptase of total RNA or mRNA (messenger RNA) is used to transcribe RNA into complementary DNA (cDNA). For further continuation of the PCR process, this cDNA is used as a template.
The Two Methods of RT-PCR
This one-step method includes a combination of reverse transcription and PCR in the same tube with a buffer. It exhibits the use of reverse transcriptase with a DNA polymerase.
In the two-step method of RT-PCR, the reverse transcription and PCR are both carried out in separate tubes. Moreover, they include the use of several optimized buffers, priming strategies, and conditions for reactions.
Elements required for RT-PCR
A single-stranded RNA extracted from the collected sample
It is an RNA-dependent DNA polymerase that uses RNA as a template to catalyze DNA synthesis.
A thermostable polymerase that can work properly at a temperature of 70 and can bear temperatures high as 98 without denaturing.
Primers are nucleic acid sequences, rather small, that start off the process of DNA synthesis.
These bases provide the energy required for polymerization and give the basic blocks needed for DNA synthesis also.
The buffer system is vital for the denaturation and renaturation of DNA. Magnesium and potassium are the most common buffers used to provide favorable conditions for that. Also, these buffers are necessary for the stability, activity, and speed of polymerase.
It is a laboratory instrument that you can utilize to heat and cool down the samples repetitively in countless cycles. The whole process helps with DNA or RNA amplification through PCR.
Steps of RT-PCR Technique
As mentioned above, the RT-PCR process is only a little different from that of PCR. Apart from the one addition of reverse transcription in RT-PCR, both techniques are almost the same. So, let’s start the RT-PCR technique from its reverse transcription stage.
A reverse transcriptase enzyme is used to synthesize a cDNA (complementary DNA) strand with nucleotides, and its end product turns out to be mRNA (cDNA hybrid). This process takes place between 40 to 50 degrees Celsius; it usually varies with the properties of the reverse transcriptase enzyme being used. Once the reverse transcription is done, the mRNAs are hydrolyzed, and during a first temperature cycle, the cDNAs are replicated by DNA polymerase.
Here, the combination of components left is heated to a temperature of 94 degrees Celsius for less than half a minute. Along with the denaturation of double-stranded cDNA into single strands, the hydrogen bonds are also broken. These single strands of cDNA then act as templates for producing new DNA strands.
This step requires an immediate decrease of temperature to 54-60 degrees for a short period of 20-40 seconds. Next, primers chain up to the DNA sequences, which starts the process of polymerization.
In this step, the DNA Taq polymerase enzyme subsequently adds bases to the end of the primer, extending the DNA sequence. It requires the temperature to be somewhere between 72-80 degrees specifically.
Each cycle results in two double-stranded DNA sequences, having one original strand and the other new-made strand in each. As the cycles carry on, these new strands also become templates after every denaturation step. With every cycle, the number of the template doubles, and like this, countless copies of the template are formed. In the end, the same fluorescence technique is also used to detect the presence of SARS-CoV-2 in this process. And that's how the Covid-19 test is considered positive or negative finally. It is also a commonly used technique in Chicago COVID testing.
Due to the intense spread of Covid-19, saving lives requires quick detection and quick cure of the viral disease. Therefore, places that do not avail proper facilities and expertise for conducting PCR tests need other immediate and amenable options. Isothermal amplification technology is one of such alternatives that are not only manageable in limited settings but has proven its quality comparable to PCR technology too.
The emerging isothermal amplification includes various potential mediums listed below:
Loop-Mediated Amplification (LAMP)
Nicking Enzyme-Assisted Reaction (NEAR)
Recombinase Polymerase Amplification (RPA)
All these mediums can be implemented in communities for the detection of the SARS-CoV-2 virus. The good thing about these mediums is you don't have to worry about sending the samples to a laboratory or getting stuck in waiting for the results of the tests, unlike the standard PCR and RT-PCR. Moreover, these isothermal mediums have been proven to be cost-effective also. They help a lot in carrying out the testing process where resources are limited and every minute costs life. Therefore, healthcare professionals worldwide need to consider these mediums if rapid control of the virus is the end goal.
Some Other Diseases Detected by NAAT
Apart from detecting the SARS-CoV-2, NAATs diagnose several other diseases also. Take malaria, for example. Along with standard polymerase chain reaction (PCR), the RT-PCR of 3 different kinds is also some of the NAATs that have been introduced to detect malaria from potential subjects. These kinds include (nested (n), quantitative (q), and real-time reverse transcription), the loop-mediated isothermal amplification (LAMP), and quantitative nucleic acid sequence-based amplification (QT-NASBA)
Besides malaria, some other diseases that NAATs diagnose include Infective Endocarditis, Genitourinary Tuberculosis, Gonorrhea, Pulmonary Tuberculosis, and many more.
Viral diseases are hazardous, and that's why their diagnosis is also a sensitive activity. While there are a lot of other ways of detecting diseases, NAAT is so far the best. It provides the highest level of sensitivity check. Although every result is always open to further interpretation, NAAT is a reliable and authentic source that ensures whatever it detects is right.
Now before you type in "rt pcr near me" into your browser, check out some of our Covid testing site locations! You may find one close to you.