bacterial identification using real time PCR - (Sep/16/2010 )
I am hopefully going to be identifying some bacterial isolates using real time PCR. I have never done this before. I was wondering if anyone can give me any guidance on this. Are there any good reviews on this I could read?
Never done it myself, but I worked in the pathogen detection lab. You say you want to "identify" them, like what kind of bacteria are there? How do you want to that by real-time, by a melting anylysis? Because usually this is done by clasic PCR and sequencing of the 16S rDNA if I remember it right. Real-time is used for quantification or detection of distinct bacteria or bacterial groups. What kind of bacteria are you looking for?
Anyway, the PCR on bacteria is tricky, especially using such sensitive methods as real-time PCR, because polymerases are produced in E. Coli and it contains traces of bacterial DNA. And there is also big problem with bacterial contamination from the enviroment, as it is a ubiquitous organism.