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

IMPORTED POST*

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

What it does well

The purpose of RT-PCR is to make a tiny amount of otherwise undetectable, but highly distinct genetic material, measurable. Once there’s more of it we can do things like check ist size is as expected, determine its exact genetic sequence or splice it in amongst other DNA. The detection part of the PCR, when used for virus identification, has changed over time. Where once we did all our analysis of the genetic material at the end of the PCR (1 or 2 hours), we can now identify if an expected generic material is present during the PCR – in real-time.

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

RT-PCR is for RNA viruses

DNA viruses include:

  • adenoviruses
  • herpesviruses
  • HIV (also has RNA stages)
  • polyomaviruses
  • bocaviruses
  • parvoviruses
  • papillomaviruses
  • poxviruses
  • megaviruses
  • many others.

PCR also works well for plasmids and human genes and other DNA fragments we want to work with in the lab.

RNA viruses include:

  • influenza viruses
  • parainfluenza viruses
  • rhinoviruses
  • enteroviruses
  • cosaviruses
  • klasseviruses
  • parechoviruses
  • respiratory syncytial virus
  • coronaviruses
  • zika virus
  • dengue viruses
  • ebolaviruses
  • human metapneumovirus
  • many others.
When high temperatures are applied by a thermocycler (the ‘oven-fridge’ machine we use to perform our PCR cycling in), any double-stranded nucleic acids fall apart or are “melted” or “denatured”.

An RT-PCR also lets us examine gene activity because it can detect and measure gene transcription products. This features the measurement of RNA not DNA. PCR won’t detect RNA without modification of the method.

How to detect RNA using a DNA amplification method

PCR is based around the use of a heat-stable DNA-dependent DNA polymerase enzyme. This enzyme was identified in bacteria that lived in hot-springs. If you put us in a hot spring, our enzymes wouldn’t keep functioning!

Make RNA into DNA

For the PCR to work with RNA as a starting material, we need to first make virus RNA genes or genomes into DNA. This provides the main PCR enzyme a template it will duplicate. Ultimately it makes enough copies to detect or use in molecular biology…or whatever the downstream application may be.

To work with RNA, we add another enzyme and a preceding step to the PCR. The new step allows us to make a DNA copy of an RNA. The enzyme is called reverse transcriptase (RT) and it’s used in the reverse transcription, or RT, step.

What’s in a name?

The addition of the RT step changes the naming of the technique from ‘PCR’ to ‘RT-PCR’ (Reverse transcription-polymerase chain reaction).

“RT-PCR” is not to be confused with real-time PCR which can be shortened to rPCR (some would argue with me on this). An RT-rPCR is used to detect and sometimes quantify RNA viruses in human samples; it’s a reverse transcription real-time polymerase chain reaction.

Methody detail

A standard PCR has about 10-30min added to it for the 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). The denaturation step intends to:

  1. knock out RT activity
  2. sometimes to activate the DNA polymerase
  3. separate any double-stranded nucleic acids into single strands (denature or melt them)

The RT step is followed by the multi-cycle PCR amplification process. This uses 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, Papadopoulos NG, Petrich A, Pfaffl MW, Rawlinson W, Reischl U, Saunders NA, Savolainen-Kopra C, Schildgen 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.

References

  1. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia
    https://science.sciencemag.org/content/230/4732/1350.long

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