From the Guidelines
Reverse transcriptase polymerase chain reaction (RT-PCR) should be used over DNA quantitative techniques like qPCR when the diagnostic target is RNA rather than DNA, particularly for detecting RNA viruses such as SARS-CoV-2, as evidenced by the high specificity and moderate sensitivity of RT-PCR in detecting SARS-CoV-2 1. This is crucial in clinical diagnostics, especially for RNA viruses, because RT-PCR can convert RNA to complementary DNA (cDNA) using reverse transcriptase enzyme before amplification, whereas standard qPCR can only amplify DNA directly. The choice between these methods depends entirely on the nucleic acid target - RNA requires RT-PCR while DNA requires standard PCR techniques. Some key points to consider when choosing between RT-PCR and qPCR include:
- The type of nucleic acid target: RNA or DNA
- The sensitivity and specificity of the test: RT-PCR has high specificity but moderate sensitivity for detecting SARS-CoV-2 1
- The clinical context: RT-PCR is necessary for detecting RNA viruses and measuring gene expression levels in conditions like cancer
- The potential for false-negative results: RT-PCR can produce false-negative results due to factors such as inadequate specimen collection, handling, and stage of infection 1 RT-PCR is also valuable for monitoring treatment response in RNA virus infections and for detecting RNA biomarkers that may indicate disease progression or therapeutic effectiveness. The fundamental difference is that RT-PCR includes an initial reverse transcription step that qPCR lacks, making RT-PCR the only viable option when RNA is the diagnostic target of interest, as supported by the most recent and highest quality study on the management of surgical patients in the emergency setting during the COVID-19 pandemic 1.
From the Research
Reverse Transcriptase PCR vs DNA Quantitative Techniques
- Reverse transcriptase polymerase chain reaction (RT-PCR) is a sensitive method for detection of RNA virus nucleic acid sequences in clinical specimens 2.
- RT-PCR is the assay of choice for COVID-19 diagnosis due to its rapidity and accuracy in informing on active coronavirus (CoV) infection 3.
- RT-PCR can detect simultaneous infections involving more than one virus, which is not possible with viral culture 2.
- Quantitative real-time RT-PCR (RT-qPCR) is the most widely used method for testing acute SARS-CoV-2 infection and is considered the "gold standard" diagnostic method 4, 5.
Advantages of RT-PCR over DNA Quantitative Techniques
- RT-PCR can detect RNA viruses, which are not detectable by DNA quantitative techniques such as quantitative polymerase chain reaction (qPCR) 2.
- RT-PCR is more sensitive than DNA quantitative techniques for detecting low levels of viral RNA 5.
- RT-PCR can be used for sample pooling, which improves the efficiency of mass screening and reduces the number of experiments performed 6.
Applications of RT-PCR
- RT-PCR is used for diagnosis of viral respiratory tract infections in children 2.
- RT-PCR is used for COVID-19 diagnosis and has been shown to be effective in detecting SARS-CoV-2 infection 3, 4, 6, 5.
- RT-PCR can be used for screening of other pathogens, such as influenza and respiratory syncytial virus 6.