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RT-PCR vs plate reader for pathogen detection


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#1 JP1

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Posted 23 July 2012 - 12:10 PM

Hi guys,

If I want to do pathogen detection, and don't care about quantification, what are the advantages of RT-PCR vs using RT-PCR chemistries in a traditional PCR machine, and measuring the result in a plate reader? It seems to me that this combination of equipment would be much cheaper than a qPCR machine, but wondering what I am missing. People always seem to do a gel with end point PCR, so not sure why fluorescent detection is not more common. Thanks for any insight!

#2 bob1

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Posted 23 July 2012 - 03:39 PM

the reason is cost - fluorescent chemistries are expensive compared to normal PCR and running a gel. Potentially you could do the experiment on a plate reader, it depends on the sensitivity of the plate reader. Fl plate readers are in the range of US$ 50,000 so about the same as a qPCR machine; filters are an extra $1000 or so usually. Diagnostic labs do this sort of thing all the time, the reason they use qPCR machines is that it allows instant detection of false postives and allows melting curve analysis too, which is very useful to make sure that the PCR is working properly.

Incidentally, RT-PCR is reverse transcriptase-PCR, you are talking about real-time PCR or qPCR, just to help you avoid confusion.

#3 JP1

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Posted 23 July 2012 - 03:58 PM

Thanks bob, I see what you are saying about cost and melting curves. What do you mean about instant detection of false positives? Is the purpose to abort the run early if the negative control starts to amplify, saving like an hour?

#4 bob1

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Posted 24 July 2012 - 02:44 PM

I mean instant compared to running a gel and then having to sequence the band or do other manipulation to find out if the amplified sequence is the correct one.




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