Realtime PCR ?!?!

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.

"The_Warrior" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]...


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