The requirement for updated and modern researches regarding the use of real time PCR in the detection of metallo-beta-lactamases in klebsiella pneumonia and pseudomonas aeruginosa...
I have had some experience on Realtime PCR. Which actually should be same in principle for different organisms as it works on RNA based quantification.
What is it actually that you are aiming at?
Are you doing direct detection from samples? If not, it's as simple as ordinary detection using primer based detection, rt master mix, no probe, non template control, and your positive control or standards, etc. You can also quantify using your standards to comparatively determine the quantity of the genes in your samples. This would be done automatically by your thermocycler, but you have to determine and insert the copy number calculated using the avocadro constant for your standards. Qualifications are only reasonable for sample based detection and less irrelevant for isolate based.
I would like to say that real time PCR (RT-PCR) is not a quantitative method at all. DNA polymerase seems to have low affinities to substrates; monomer enzyme has Km values c.a. 50 μM to dNTPs, but dimer biotinidase has Km 0.48 μM to biocytin (please see files; BIN Mr 110,000 and The Fascio Effect). Further, direct use of fluorimeter without purification is not quantitative due to violence against the Lambert-Beer's law.
I have instead searched for Klebsiella spp on my protein database made by PDMD (Protein-Direct-Microsequencing-Deciphering) method (please see file; HepG2 fucoidan). PDMD method can also detect proteins of invaded microbes.
Only two specimens has proteins of Klebsiella spp.
Serum of 12mo (female, common cold) has Homoserine kinase (Klebsiella pneumoniae) at 5.9 μg/mg of serum protein. Klebsiella pneumoniae or its bacteriophage seems specifically to induce Insulin-like growth factor 1A precursor/IGF-I precursor (9 - 118 or 195) at 6.7 μg/mg of serum protein.
A bovine milk has Dihydrofolate reductase (Klebsiella aerogenes/Enterobacter aerogenes) at 10.7 μg/mg of milk protein.
PDMD method usually gives quantitative results.
Dear Kou Hayakawa
Thank you very much for your valuable interaction and so interesting attached file. Regard
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