RTPCR Test is the most reliable laboratory method for detecting COVID-19 Virus. This post will discuss what is RTPCR Test, how it detects Covid-19 Corona Virus, what is CT value, meaning of its test results and advantages of this test.
What is RTPCR Test
RTPCR Test stands for Reverse Transcription Polymerase Chain Reaction Test also called as Real Time PCR Test. It is one of the most reliable and fastest laboratory methods for detecting and tracking COVID-19 Virus. This test is also used in the study of this Virus. This test was also used in the detection of Ebola virus and Zika virus.
Fig. 1 – Introduction to RTPCR Test
This test basically detects the presence of specific genetic material in disease causing Microbes also called Pathogens or Virus. The genetic material is either DNA (Deoxyribonucleic acid) or RNA (Ribo Nucleic Acid). Covid-19 Virus contains only RNA and the detection of this Virus includes the conversion of RNA to DNA which is also called as Reverse Transcription.
RTPCR Test includes creating complementary DNA (cDNA) by reverse transcribing RNA template using Reverse Transcriptase (RT) enzyme. This enzyme uses the RNA template to produce a complementary single-stranded DNA called cDNA.
Further, DNA polymerase enzyme converts the single-stranded DNA molecule into double-stranded DNA which can be used as templates for a PCR reaction. This helps DNA to be copied and amplified exponentially with the use of primers and thermocycling . The value of RTPCR or simply CT value helps to determine if a person under test has Covid-19 Virus or not.
Fig. 2 – Reliable Test for Covid-19
How does COVID-19 RTPCR Test Work
There are four important steps involved in RTPCR Test. They are:
- Sample Collection
- Inactivation of Virus
- Extraction of RNA
- RTPCR Testing
This step involves collection of nasal swab. This requires collection of test sample of Nasal or respiratory secretions from back of the nose and throat. This sample is sent to the laboratory for further testing.
Inactivation of Virus
Inactivation of virus is an important step to inhibit viral replication and contamination. This is done by altering the environment around the Virus through heating, pH treatment, UV light treatment or solvent/ detergent treatment etc. The inactivated samples are then subjected to Extraction.
Fig. 3 – PCR Machine/ Thermal Cycler for RTPCR Test
Extraction of RNA
From the inactivated biological sample, RNA is isolated and purified. This is done using commercially available RNA extraction kits or through organic extraction. This process removes proteins, fats etc. from the sample. The obtained RNA sample is stored in RNase free water.
RTPCR (Reverse Transcription Polymerase Chain Reaction) testing involves two steps:
- Reverse Transcription
- Polymerase Chain Reaction
It involves conversion of RNA into Complementary DNA (cDNA) using specific enzymes. Reverse Transcriptase is an enzyme used in the synthesis of DNA molecule. This DNA is called as Complementary DNA which is used as a template for PCR Testing in RTPCR Test.
Polymerase Chain Reaction
This process involves amplifying DNA sequence to millions of copies and this is done using an equipment called as Thermal cycler. The sample is placed in the sample well and illuminated with a tungsten halogen lamp. Light passes through five excitation filters before reaching sample wells. Includes detection and monitoring of the process of amplification in real time using fluorescence.
The intensity of fluorescence signal is directly proportional to the copies of DNA being produced. If the fluorescence intensity crosses certain threshold, then the test is determined positive. If the virus is not present in the sample, then PCR test of RTPCR test procedure does not make copies of DNA and thus can conclude if the sample under test has Corona Virus strains or not.
Fig. 4 – RTPCR Test Procedure
What is CT Value
CT Value or Cycle Threshold value is a measure of how soon the Virus gets detected in a sample under test. Patients with CT (Cycle Threshold) value less than 35 may be considered positive, while those with a CT value greater than 35 may be considered negative. Samples with a CT value equal to 35, which is seen as a poor sigmoidal curve, should be essentially retested.
- Lower CT Value = Higher Viral load
- Higher CT Value = Lower Viral load
What do Covid-19 RTPCR Test Results Mean
Whether the patient is positive or negative, the RTPCR test is only indicative of whether the virus was present at the time of test. It neither rules out whether the patient was infected in the past and therefore has developed immunity, nor that the patient is at an early stage and would show symptoms in the near future.
Covid-19 test results can be either:
If a person is Covid positive, he can be in home isolation with a watch on his/her health under medications. If the conditions get severe, he could visit medical practitioner or nearby hospital.
If a person is Covid negative, it is still advisable to maintain social distancing with a mask on and to stay at home to protect yourselves and others.
Advantages of RTPCR Test
The advantages include:
- Currently, it is the most reliable testing technique.
- Minimally invasive test.
- Results are accurate.
- Carry over contamination is avoided.
- Analysis is fast and easy.
Disadvantages of RTPCR Test
The disadvantages are:
- Sometimes this test produces false positive reports when the person under test has common cold.
- People might find it uncomfortable when they are asked for a nasal swab.
- Sample must be pure without contamination else the results are not accurate.
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