Step 2 – Primer Annealing: Next, is the addition of short fragments of DNA, called primers. This is accomplished by heating the DNA to high temperatures (> 90☌) for amount 10 min. Thus, the next step is to unwind the DNA molecule into separate DNA stands. Step 1 – Denaturation/Separation: To begin, it is important to remember that DNA is a double-stranded structure. This includes human RNA, bacterial RNA, even other coronavirus RNA and if present, SARS-CoV-2’s RNA. Thus, the role of reverse transcriptase inside the reaction mixture is to convert all the RNA present within a given sample into cDNA. Reverse Transcription: As mentioned earlier, PCR only works on DNA templates. The ingredients inside this mixture includes DNA polymerase, reverse transcriptase, DNA building blocks, and specific fluorophore probes and primers that recognize SARS-CoV-2. Next, the RNA sample is added into one reaction mixture containing all the ingredients required to complete the diagnostic test, also known as “one-step RT-PCR”. Sample Preparation: Once the specimens arrive at the laboratory, researchers will use available commercial purification kits to extract RNA from the sample. The sample is then placed into a sterile tube containing viral transport media to keep the virus viable (3). Sample Collection: To start the diagnostic test, a trained healthcare worker will use a swab to collect nasopharyngeal specimens from the patient’s Altogether, these features contribute to the versatility and sensitivity of Real-Time RT-PCR as a diagnostic test for infectious diseases. genetic material of SARS-CoV-2), researchers have to use a special enzyme – called Reverse Transcriptase – to convert RNA into DNA templates, also known as complementary DNA (cDNA). Thus, in order to apply Real-Time PCR on RNA samples (i.e. A major limitation of PCR is that it detects only DNA templates. By measuring this fluorescence intensity, one can quantify the amount of genetic material inside the sample. To visualize the amplification of DNA, Real-time PCR uses increases in the fluorescence intensity of a fluorogenic probe in proportion to the amount of amplified DNA. This technology was later improved to allow “real-time” visualization and quantification of DNA targets as they undergo amplification. Mullis, in the 1980s, to allow researchers to amplify and detect specific DNA targets (1,2). The foundation of Real-Time RT-PCR derives from Polymerase Chain Reaction (PCR) a laboratory technique developed by Nobel Prize-winner, Kary B. This genetic material can be specific to humans, bacteria, and viruses like SARS-CoV-2. Real-Time RT-PCR (Reverse Transcription Polymerase Chain Reaction) is a sensitive and fast test used for detecting the presence of specific genetic materials within a sample. Furthermore, as long as the virus’ genetic material is present inside the cell, we can use a laboratory technique called Real-Time RT-PCR to determine whether a patient has been/is infected with SARS-CoV-2. Similar to other viruses, when SARS-COV-2 infiltrates a cell, it releases its RNA and exploits the cell’s machinery for viral replication. Consequently, viruses like SARS-COV-2 are required to invade healthy cells in order to survive and multiply. It is also important to note that SARS-CoV-2 contains only RNA.Ī common feature among all viruses is that they depend on host proteins and reproductive machinery for survival. In terms of diagnostics, DNA is more stable for testing infectious disease than RNA because of its structural and intrinsic properties. The major difference between the two types of genetic materials is that DNA is a double-stranded structure whereas RNA is single-stranded. There are two types of genetic materials: Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA). Genetic material is the instruction manual within a cell or virus that provides the directions on how to behave, survive and more. What is a genetic material and how is it used to test for infectious diseases? It can be of value to public health practitioners or anyone in healthcare community involved in the COVID-19 response. It also provides the basics on interpreting test results that can be helpful in understanding the state of the disease and/or its progression and the likelihood of transmissibility. It provides basic information on how this molecular biology test is used in laboratories to detect genetic material of a pathogen such as SARS-CO-2 virus, the cause of COVID-19 disease. This document provides a brief description of the Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) test. COVID-19 Real-Time RT-PCR diagnostic tests can provide information on whether or not a patient has been infected with SARS-CoV-2 by detecting and measuring the virus’ genetic material.
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