Realtime PCR ?!?!

N10 limbic_lesion at hotmail.com
Thu Mar 10 06:01:24 EST 2005


"The_Warrior" <almahdi at magma.ca> wrote in message 
news:jMmdndn5RLZK7bLfRVn-ug at magma.ca...
> Hello everyone, I would like to know what you guys think about the 
> following
> techniques in terms of their efficiency, mechanisms of action, and 
> accuracy
> in identifying bacterial species. Also, if you have some references about
> these techniques, please send me a copy of the references.
>
>
>
> - PCR
>
> - Real Time PCR
>
> - Cytotoxin assays
>
>
>
> Thanks everyone.
>
>


Hi Warrior

Microbial identification can take altleast two paths  which can converge 
depeding on the nature of the organisms you are dealing with.

Firstly consider a completely unknown isolate or isolates growing  on an 
agar plate.  Firstly you need to produce prue colonies and then begin the 
task of identification. PCR probes of any kind are highly specific  and 
therefore are tools to "ablosutely " confirm a suspicion of identity  or 
not. They generally are a single shot stab at verification.

For example if I isolate an H2S postive  gram negative  rod on XLD agar 
there is a reasonable probability that it is Salmonella  but equally it 
might be Proteus or some other memeber of the Enterobacteriaceae family . So 
lest say i go ahead and invest  the best part of a day extracting DNA , 
geeting rid of  inhibitors and run either PCR or rtPCT  with a SAlmonella 
probe  and the result is negative !. GAsp Im no furhter forward  so Im 1 
genus down  15 more to go. Ofcourse I could  have run a mutiple array of 
probes ( one for each genus in the Enterobacteriaceae). Now im approaching 
obtaining a usefull result an rrayof negtaives andone postive. If I find my 
isolate is Salmonella the question arises which SAlmonella  so I begin 
looking for other probes to  characterisethis beast to species level.

ALl quite uncessary, huge effort  and excessively expensive inthe situation 
where one is looking at an UNKNOWN on a plate. What if you qwere lokking at 
20 unknows  isolated on none selective agar. Such a task would need a 
massive array of probes ...do such exist ..I dont know.

In a dealing with UNknown organisms its perhapes quicker in the long run, 
less expensive and more immediate ( more fun)  to go the other route of 
classical identification.

Gram + MIscoscopy+ ancillary stains
Motlity
Oxidase catalayse
Fermentation pattern
Key generic  identifiers ( eg Lysine decaboxylase  Lecintinase production 
etc etc )
Cytotoxin production (obviuosly only for toxin competent strains)

If your labour of love turns out to be that special organism your can always 
go ahead and  ultimately confirm with PCR if a probe exists.

Its a  different emphasis in a mass screening situtaion for example   where 
the vital out come is to know whether or not a partical organism or 
organisms  are present in a particular matrix or sample type. Say screening 
a population of humans for infection by  some  virus. Here the YEs no answer 
is sufficient and in fact a negative is a good result . PCr and rtPCr are 
ideal tools for such a task  in terms of lab efficiency and accuracy of 
outcome.


In my own area of work I use rtPCR screening of food to detect Salmonella, 
Listeria and  Ecoli 0157 H7. Im looking for negatives, hundreds of them and 
perhapes the occasional positive which will be go on to be confirmed by 
conventional isolation  and identification technique.

So you see the apporach is to a large degree dictated by the task at had and 
what out come or empahsis  you wish to achieve.

I d say to you as a student if you have the oppurtunity to work with PCR 
and become competanmt and knoweledglable in its practise and theory ..go for 
it . Design your project around the technique . The reaction was truelly a 
Damacus MOment for the guy who conceived it  ( Look it up in Scientific 
American) , stroke of mental genus which will impact upon biological 
sciences for decades and decades. PCR is the key to the chemical box of 
chocolates

Do well

Best N10






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