How is the RT-PCR Test Done

The RT-PCR test is an accurate test for diagnosing infectious diseases, especially COVID-19. The acronym stands for reverse transcription polymerase chain reaction. This test has become immensely popular since the emergence of the Coronavirus pandemic. A positive test is a reliable indication that you suffer from COVID-19. On the other hand, a negative test probably means good news—that you have been spared this disease. Read this article to know exactly how the RT-PCR is done and what you can expect.

What is the RT-PCR test?

Before learning how the RT-PCR is done, you must first know what it is. A PCR (polymerase chain reaction) test is a quick and precise way to diagnose genetic variations and infectious diseases, most prominently COVID-19. The basic premise behind the working of this test is to locate the DNA or RNA of a pathogen that causes the disease. The test can also discover abnormal cells in a sample to diagnose a disease.

The test facilitates the detection of genetic material from a particular organism, usually a virus. This is how RT-PCR is done. The presence of a virus can easily be detected with this test if the person undergoing it had the virus at the time of the test. Medical experts can also identify virus fragments even after the patient has recovered from this test.

Who should get tested for COVID-19?

As we have already learned, this test can detect infectious diseases like COVID-19. So, the experts recommend this test if you suffer from the following symptoms:

  • Cough
  • Shortness of breath
  • Fatigue
  • Diarrhoea
  • Runny nose
  • Muscle pain
  • Fever
  • Sore throat
  • Headache
  • Loss of taste
  • Loss of smell Congestion
  • Nausea or vomiting
  • Congestion
  • Nausea or vomiting

How does RT-PCR detect COVID-19

The SARS-CoV-2 virus is the culprit agent behind COVID-19. It is an RNA virus that multiplies and survives in a healthy cell after infiltrating it. The SARS-CoV-2 RNA can be accurately detected with the help of an RT-PCR test. In this test, the conversion of the RNA takes place to DNA via the ‘reverse transcription’ process. This conversion is the key to the detection of viruses.
Medical experts detect SARS-CoV-2 RNA in the respiratory specimens when the patient is in the infection’s acute phase. Samples are collected for various respiratory regions like:

  • Nasal
  • Nasopharyngeal swabs
  • Sputum
  • Lower respiratory tract aspirates
  • Bronchoalveolar lavage
  • Nasopharyngeal wash/aspirate or nasal aspirate

Experts treat the collected sample with chemical solutions to remove certain unwanted substances. After collection, the expert extracts only the RNA from the collected sample.

How is the RT-PCR done?

The question ‘how is RT-PCR done’ can be answered in the following three key steps:

Sample collection

Here, a healthcare provider collects respiratory material from the nose by inserting a swab. A swab is a soft tip on a flexible and long stick.

Nasal swabs and nasopharyngeal swabs are the two types of swabs used. The former collects nostrils samples while the latter collects deeper nasal cavity samples. Both types are suitable for collecting material. After collection, the swab is sealed in a tube, which is sent to a laboratory for further testing.

Extraction

The swab is sent to a laboratory in a sealed tube after collection. After receiving the sample, the laboratory scientist isolates or extracts the genetic material from the rest of the sample material.

PCR

In this step, special chemicals are used along with a PCR machine. Here, the amount of the targeted genetic material is amplified due to the hot and cold cycle. After plenty of cycles, several copies of a small portion of the SARS-CoV-2 genetic material are created.

One of the tube chemicals results in the production of fluorescent light in case there is SARS-CoV-2 in the sample. When the amplification becomes significant enough, this signal can be detected by the PCR machine. Scientists use this special software to interpret the signal as a positive test result.

Method of RT-PCR testing

There are two different ways to get a test sample. The method Of RT-PCR testing can take two forms—blood test and nasal swab.

Blood test

Here, a health care professional extracts blood from your body by inserting a small needle into your vein. When the needle is inserted, a small amount of blood from the body is collected in a vial or test tube. This small amount of blood serves as the sample. The whole procedure takes just a few minutes, and you may feel a little pain when the needle is inserted.

Nasal swab

Here, samples are collected from the nostrils by a healthcare expert. The samples can be collected either from the front or the back of your nostrils. The samples may also be extracted from the nasopharynx, which is the upper part of the throat/nose. This is the most common way of conducting RT-PCR testing.

The procedure of the nasal swab method of RT-PCR testing is as follows:

  • The healthcare professional will tilt your head back.
  • Then, the expert will insert a swab inside the nostril gently.
  • The swab will be rotated inside the nostril.
  • The swab must be left inside for 10 to 15 seconds.
  • The swab is removed after 10 to 15 seconds, and the process is repeated in the other nostril.

How do the test results arrive?

A real-time RT-PCR setup has to undergo 35 cycles as per the standard procedure. As such, around 35 billion new copies of the viral DNA sections will be created. These copies pertain to each sample virus strand.

As new copies are created, marker labels attach to the DNA strands. Afterwards, a fluorescent dye is released. The machine’s computer measures this dye, and you can see this on the screen in real-time.

The computer tracks the amount of sample fluorescence after each cycle. When this amount exceeds a certain level of fluorescence, the presence of the virus is confirmed.

Scientists may also monitor how quickly this level is reached, that is, how many cycles it takes to reach this level. The fewer cycles it takes, the more severe the disease.

What is the meaning of the RT-PCR result?

A positive test result means a significant likelihood that the person is affected by Coronavirus. Sometimes, a person may be asymptomatic and display no symptoms of COVID-19, even after appearing positive on the test.

People who test positive may display only mild symptoms in some cases. In such cases, patients can recover safely in their homes without being admitted to a hospital. However, if the symptoms become severe, the person must immediately contact a healthcare provider.

A negative test result means a considerable likelihood that the person is not affected by Coronavirus. However, it could also mean that the person did not have the virus when the test was taken. So, if people receive negative test results, it does not necessarily mean that the test was ineffective.

This test is not perfect, and it is possible that the test was unable to detect the Coronavirus. So, as pointed out by clinical experts, a negative test is not a guarantee that a person is safe.

The person may still be exposed to the Coronavirus after undergoing the test and may spread the virus. As such, one must continue to take protective steps even after receiving negative RT-PCR test results.

How long after being infected do you test positive for COVID-19?

Owing to the RT-PCR test’s immense sensitivity, detecting extremely small virus material amounts is possible. This means fragments of the Coronavirus can be detected even after the patient has recovered from COVID-19 and is no longer contagious. People may test positive for a while after they contract the illness.

People whose immune systems have been compromised may experience prolonged sickness. Healthy individuals may also be re-infected by the virus.

Difference between the PCR and antigen tests

There are two types of tests for the detection of COVID-19.

Polymerase chain reaction (PCR): This detects the presence of the virus’s genetic material. The test also identifies the presence of the virus fragments. According to most healthcare experts, this is the most accurate and reliable test we have. The performance of this test can go on for hours.

Antigen test: This detects protein bits on antigens’ surfaces. Antigens are a type of virus. The test’s performance duration ranges from 15 minutes to half an hour. They are highly accurate but only within the first few days of the onset of symptoms.

Where to get tested?

If you display COVID-19 symptoms or were around positively tested people, get yourself tested immediately. Also, talk to your doctor about your condition and the possible consequences.

Your doctor will provide you with certain instructions after reviewing your symptoms or circumstances. This could be done either face to face or via online or a video call. If necessary, the doctor will recommend that you get a test done. The doctor may suggest a testing facility or may help you find one.

Make sure to communicate with the facility, if possible, to make preparations beforehand. You can also learn about the details of testing facilities by visiting websites on the internet.

What to expect from this test

A sample of your mucus or saliva shall be taken from your nose or throat by a healthcare expert. This is required for diagnostic testing. The collection may happen at a healthcare facility like a hospital, a doctor’s private clinic, or at a drive-up testing centre.

Professionals from home service providers such as MyGate can also be invited to a place of your convenience for sample collection.

For the nose swab, a healthcare professional will insert the cotton tip of a thin stick inside your nose. This is in order to collect a mucous sample. You may feel a slight discomfort, but it’s not a big deal.

A saliva sample is comparatively easier and more comfortable. All you have to do is spit in a tube multiple times. This way, a sample of your saliva gets collected for laboratory analysis.

If there is a need for a blood test, then expect a needle to be inserted into a part of your body, usually the arm. You may feel slight pain during this insertion but not to the degree to make you concerned.

Accuracy of the RT-PCR results

The results of the RT-PCR are not 100% accurate. False positives and negative incidents have been reported across the world. Positive results may be an indication of Coronavirus but may not entirely be accurate.

However, this does not mean that you start doubting this test. Despite not being completely precise, the test is still quite reliable. In fact, this is the best testing method we have so far to detect COVID-19.

Pros of RT-PCR testing

RT-PCR testing is an accurate and reliable test that has the following pros:

Minimally invasive– You can have this test performed in a minimally invasive manner by using nasal swabs.

Possibility of isolation– The RT-PCR can sometimes be taken from the car or home of the patient, thereby facilitating proper social distancing.

Reduced false negatives cases– Nasal swabs, especially deep ones, will have fewer false negatives compared to other tests.

Cons of RT-PCR testing

The method of RT-PCR testing is not entirely flawless. Below are the various cons of RT-PCR testing:

Long wait for the result– Sometimes, RT-PCR test results can take up to 1 to 2 weeks to be reported. This was the reality during the COVID-19 pandemic.

False negatives– The molecular tests have occasionally failed to detect the virus when the patient actually did have the virus. The ranging of the false positives rate is from 2 to 37 per cent.

Discomfort– Deep nasal swabs and needle insertions can be uncomfortable for children and older adults.

COVID

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