PCR amplification using universal 16s rDNA - it's necessary to do it (Apr/17/2007 )
I do PCR for the detection of human cariogenic bacteria, my samples are dental plaque (clinical samples), I have done PCR detection of bacteria at the species level (Streptococus mutans), all my partners in the lab are using universal 16s rDNA for the detection of the bacteria in the enviroenmental samples, then they use primers to detect different types of bacteria at the genus level. Do I have to do the universal 16srDNA amplification? the primers that I use are for sure specific to bacteria species, so I thought that there is no need to use 16s rDNA, in addition to that my samples are clinical samples......, Could some one help me and answer my question and give me an explanation
This depends on your goal. Do you need to survey all organisms present, or do you merely need to know whether specific organisms are present? How precisely do you need to identify these organisms? Your goals will set the strategy.
I need to know the presence of specific organisms, so what I have to do the 16s rDNA rxn or not?
You can probably use your primers directly. The potential problem is lack of specificity of your primers. Your colleagues may be doing nested PCR, in which the 16S rRNA region from all sample is amplified with universal primers, followed by reamplification with species specific primers using the pooled 16S sequences as a template. If you directly use your species specific primers, you may amplify something in the clinical sample which does not come from the 16S region. This is unlikely, but could happen with less than perfect primers.