Dear Dr. Zimmermann,

Thank you so much for your answer

Actually, if it's a chromosomic gene why we don't detect nanC gene in all pneumococcal strains using a conventionnal PCR?

There are methods/ commercial kits to isolate plasmid DNA. Than just run your PCR on such plasmid DNA.

Jan Tkadlec Thank you Jan, which kit would you recommand ?

The kit that came to my mind is QIAGEN Plasmid Mini Kit. However, I never used it for what you are going to try. So I suggest that you have to use some controls to ensure that the plasmid isolate is free of chromosomal DNA. My recomendation is to made two separate isolations: i) to isolate chromosomal DNA from the strain ii) to isolate plasmid DNA using the kit. Than you have to detect some chromosomal gene that is in the genome of your bacteria. For chromosomal DNA the result should be positive and for plasmid DNA isolate it should be negative - meaning that plasmid DNA is free of the bacterial genome. then if you will detect in the plasmid DNA your gene of interest it is highly probable that it is located on plasmid.

Stefan Zimmermann

thanks Dr. Stefan

Actually, i tested around 20 to 30 of invasive strains and an important number of non invasive strains each month. But, we detect the presence of the gene nanC for some of them and the absence for others using PCR.

best regards

asma

Jan Tkadlec

Thank you so much Dr, Jan