PCR-related techniques for detecting

Juck, David juck at biotech.lan.nrc.ca
Tue Apr 4 10:47:58 EST 1995


>How about devising a system based on detecting RNA? This is degraded 
>faster than DNA, so you'd have less chance of false positives based on 
>nucleic acids from dead cells.

>Another alternative approach would be to follow up any PCR positives 
>with traditional plating-out, with a positive result in both required for 
>confirmation of the presence of the pathogen in food. There would still be 
>an advantage in using PCR because it would screen out the negative 
>samples.

>No references for either of these - just a couple of ideas that occured to 
>me over my mid-morning cuppa:-) 

>Kevin O'Donnell
>Scottish Agricultural Science Agency    
>Edinburgh
>Scotland                                           

With respect to detecting RNA using PCR, there is a commercially available 
kit for RT-PCR (reverse transcriptase-PCR) produced by Perkin-Elmer which 
first produces a DNA template from the RNA in the sample, and then normal PCR 
is performed on the DNA template. This should be a good indicator of 
metabolic activity of the cell, but if the cell is in a dormant state, false 
negatives may be a factor. Choice of the target sequence then becomes 
important. I have not used RT-PCR, but someone in my lab is currently using 
it in soils and is getting interesting results.

With plating out of the samples, the same problem is possible with dormant 
cells not forming colonies. After an incubation time, cells may become active 
and start to reproduce. I don't mean to play the devil's advocate, this may 
not be a problem on foods, but in water monitoring, it can be an important 
factor.

David Juck
Biotechnology Research Institute
Montreal, 
Canada.



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