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PCR stands for polymerase chain reaction, a laboratory method used to amplify specific regions of DNA for diagnosis and analysis in medical research. RT-PCR works on this principle of reverse transcription. He won the Nobel Prize in Chemistry for introducing this technique and revolutionizing the field of molecular biology. It became the benchmark technology for detection of specific RNA sequences and it was because of this sensitive in vitro method that mass diagnosis of coronavirus became a possibility.

This cDNA then undergoes exponential amplification using PCR to form multiple copies, which are then used for downstream analysis. However, in order to amplify RNA, reverse transcription needs to be carried out since RNA is not an efficient template for Taq polymerase.

In this method, the reaction tube of reverse transcription and polymerase chain reaction is the same, and both processes are performed simultaneously. This simple and convenient one-step approach is used for performing a small number of assays. Since both reactions are conducted in a single reaction tube, the primers used are sequence-specific.

The reaction vessel, buffer, reagents, conditions, and priming strategies used in each step are different. This allows a higher yield of cDNA to be obtained which can either be stored or further amplified. It is a quantitative method of analysis using the principle of a typical PCR. In this process, however, the measurement of DNA amplification is carried out in real-time instead of at the end of the process with an agarose gel. Quantitation of PCR products is performed using fluorescent probes like intercalating dye or hydrolysis-based fluorescent probes.

Quantitative PCR helps in the detection of pathogens and the determination of the copy number of the DNA sequence of interest. Moreover, model systems with inhibitors, stimulants, siRNA, or knock models are used to investigate gene expression changes. Quantitation and data analysis of the results from cycles of qPCR and RT-qPCR are performed with the help of an amplification curve with initiation, exponential, and plateau phases.

This amplification curve is generated using a serial dilution of standards of known concentration. The analysis of the data can be based on absolute quantitation or relative quantitation. This method has given new hope to medical research for conducting disease diagnostics, molecular cloning, and recombinant DNA technology. RT-PCR requires a proper setup and contamination-free environment for its conduction. A life science or medical laboratory is incomplete without a high-quality PCR setup.

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Why does rt pcr take time - why does rt pcr take time

 

Importantly, the internal control should be included to help identify the specimens containing substances that may interfere with the extraction of nucleic acid and PCR amplification. Because of the several risks to patients in the event of a false-positive result, all clinical laboratories using this test must follow the standard confirmatory testing and reporting guidelines based on their proper public health authorities.

In conclusion, according to the mentioned reasons, the results of real-time RT-PCR tests must be cautiously interpreted. In the case of real-time RT-PCR negative result with clinical features suspicion for COVID, especially when only upper respiratory tract samples were tested, multiple sample types in different time points, including from the lower respiratory tract if possible, should be tested.

Proper sampling procedures, good laboratory practice standard, and using high-quality extraction and real-time RT-PCR kit could improve the approach and reduce inaccurate results. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Peer reviewers on this manuscript have no relevant financial relationships or otherwise to disclose. Expert Rev Mol Diagn.

Published online Apr Alireza Tahamtan a and Abdollah Ardebili b. Author information Article notes Copyright and License information Disclaimer. Received Mar 14; Accepted Apr This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source.

These permissions are granted for the duration of the COVID pandemic or until permissions are revoked in writing. This article has been cited by other articles in PMC. Associated Data Data Citations [[cited Mar 23;]]. Expert opinion In conclusion, according to the mentioned reasons, the results of real-time RT-PCR tests must be cautiously interpreted. Funding Statement This paper was not funded.

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. Reviewers disclosure Peer reviewers on this manuscript have no relevant financial relationships or otherwise to disclose. References 1. Recent advances and perspectives of nucleic acid detection for coronavirus.

J Pharm Anal. DOI: Int J Mol Sci. November 23; 17 11 :E Simultaneous detection of severe acute respiratory syndrome, middle east respiratory syndrome, and related bat coronaviruses by real-time reverse transcription PCR. Arch Virol. J Med Virol. February 25 [Epub ahead of print] DOI: Understanding the influence factors in viral nucleic acid test of novel coronavirus nCoV.

Chin J Lab Med. Phan T. Infect Genet Evol. February 21; 81 Clin Infect Dis. 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 It would be easier to follow up and understand the whole step-by-step detection process via NAAT this way. 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, it is the upper or lower respiratory tract. It can also be done with a saliva specimen, but their quality tends to vary highly. 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 , 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 strongest sensitivity level, as checked and confirmed, is that of laboratory-based NAATs. Therefore, laboratory-based NAATs are trusted more and considered more authentic.

The many different methods of NAATs used to amplify nucleic acids and detect the virus causing Covid It is considered a standard test to diagnose the fatal Covid 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. 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. Although most of these tests take hours to be done, some of them are faster too. But do you know what it really is? Well, this particular technique is a process carried out inside a laboratory. 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.

Both of the methods have a slight difference. 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 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. Moreover, they include the use of several optimized buffers, priming strategies, and conditions for reactions. DNA Polymerase A thermostable polymerase that can work properly at a temperature of 70 and can bear temperatures high as 98 without denaturing. Primers 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.