Reverse transcription polymerase chain reaction (RT-PCR)…a primer for virus detection

IMPORTED POST*

The polymerase chain reaction (PCR) is a cyclical enzyme-driven amplification technique for copying a chain of DNA into billions of new copies.

The purpose of PCR is to make a very tiny amount of very distinct genetic material detectable and to use some form of technology to detect what would otherwise be too little material to see in the first place.

The process of PCR is covered on the PCR page so I won’t repeat it all here.

Before we use PCR for virus detection from human samples, we usually prepare the nucleic acids with an extraction or purification step. This process gets rid of proteins and carbohydrates and inhibitors of the PCR leaving a fairly pure nucleic acid preparation. The shorter this purification process is, the better for faster testing of a lot of samples.

PCR can be used to detect some viruses ‘as is’. These viruses have genes or a genome that is made of DNA. But many viruses don’t store their genetic code as DNA, they use RNA as the plan from which they make more of themselves and their viral proteins. In these cases, DNA only plays an intermediate role, if any. 

DNA viruses include the adenoviruses, herpesviruses, HIV (an example which also has RNA stages), polyomaviruses, bocaviruses, parvoviruses, papillomaviruses, poxviruses, megaviruses and many others. PCR also works well for plasmids and human genes and other DNA fragments we want to work in the lab for reasons other than detecting a virus in a human.

RNA viruses include influenza viruses, parainfluenza viruses, rhinoviruses, enteroviruses, cosavirus, klasseviruses, parechoviruses, respiratory syncytial virus, coronaviruses, zika virus, dengue viruses, Ebola virus, human metapneumovirus and many others.

When high temperatures are applied by a thermocycler (the ‘oven-fridge’ we use to perform our PCR cycling with), any double-stranded nucleic acids fall apart or are denatured.

We also look at gene activity which involves detecting and measuring gene/genome transcription which also features the measurement of RNA not DNA. PCR won’t detect RNA without modification of the method.

PCR is based around the use of a heat stable DNA-dependent DNA polymerase enzyme. For the PCR to work, we need to first make virus RNA genes or genomes into DNA so that the main enzyme can use it and duplicate it and make enough of it to detect or use in molecular biology…or whatever the downstream application may be.

To make a DNA copy of the RNA, we need to add in another enzyme and another, preceding, step to the PCR process. That enzyme, the reverse transcriptase (RT) is added in a step called reverse transcription (also RT!).

The addition of the RT step changes the naming of the technique from ‘PCR’ to ‘RT-PCR’.

RT-PCR is not to be confused with real-time PCR which is shortened to rtPCR (many would argue with me on this). So an RT-rtPCR, which we use when detecting and sometimes quantifying RNA viruses in human samples, is a reverse transcription real-time polymerase chain reaction.

A standard PCR has about 10-30min added to it for this new step. The new incubation happens at an appropriate temperature (40-50°C) and is followed by a denaturation step (92-95°C for 2-15min) to:

  1. knock out RT activity
  2. sometimes to activate the DNA polymerase
  3. separate any nucleic acid that is double-stranded

The RT step is then followed by the multi-cycle PCR amplification process which can now use the new DNA strand as a template for exponential copying….the billion-fold amplification reaction, or ‘PCR’ part.

Further reading..

  1. PCR primers…a primer!
    http://virologydownunder.blogspot.com.au/2015/05/pcr-primersa-primer.html
  2. The mechanics of the polymerase chain reaction (PCR)…a primer
    http://virologydownunder.blogspot.com.au/2015/05/the-mechanics-of-polymerase-chain.html
  3. Mackay IM. Real-time PCR in the microbiology laboratory. 2004. Clin Microbiol Infect. 10(3):190-212.
  4. Mackay IM, Arden KE and Nitsche A. 2002. Real-time PCR in virology. Nucleic Acids Res. 30;6. 1292-1305. 
  5. Beld MGHM, Birch C, Cane PA, Carman W, Claas ECJ, Clewley JP, Domingo J, Druce J, Escarmis C, Fouchier RAM, Foulongne V, Ison MG, Jennings LC, Kaltenboeck B, Kay ID, Kubista M, Landt O, Mackay IM, Mackay J, Niesters HGM, Nissen MD, Palladino S, Papadopoulous NG, Petrich A, Pfaffl MW, Rawlinson W, Reischl U, Saunders NA, Savolainen-Kopra C, Schoildgen O, Scott GM, Segondy M, Seibl R, Sloots TP, Wang Y-W, Tellier R and Woo PCYl. Chapter 10:”Experts’ roundtable: Real-time PCR and microbiology”, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
  6. Mackay IM, Arden KE, Nissen MD and Sloots TP. Chapter 8. “Challenges facing real-time PCR characterisation of acute respiratory tract infections”, In: Real-Time PCR in Microbiology, Mackay IM (Editor). 2007. Caister Academic Press, Norfolk, UK. 269-317.
  7. Mackay IM, Mackay JF, Nissen MD and Sloots TP. Chapter 1: ”Real-time PCR; History and fluorogenic chemistries”, In: Real-Time PCR in Microbiology, IM Mackay (Editor) 2007. Caister Academic Press, Norfolk, UK.
  8. Mackay IM, Bustin S, Andrade JM, Kubista M and Sloots TP. Chapter 5:”Quantification of microorganisms: not human, not simple, not quick”, In: Real-Time PCR in Microbiology, IM Mackay (Editor). 2007. Caister Academic Press, Norfolk, UK.
  9. Mackay IM, Arden KE and Nitsche A. Real-time fluorescent PCR techniques to study microbial-host interactions. Methods in Microbiology, Microbial Imaging. (2005) Vol 34. Chapter 10.Elsevier. pp255-330.
  10. Mackay IM. Respiratory viruses and the PCR revolution. In: PCR Revolution: Basic technologies and applications, Bustin, SA (Editor). 2010. Ch 12. Pp189-211. Cambridge University Press.
  • *IMPORTED POST
    05MAY2015. This post was posted over on my old blog platform virologydownunder.blogspot.com.au and has now been moved to here. 

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