I am using nested PCR to diagnose Candidatus neoehrlichia mikurensis 16S rRNA gene and I got bright bands at the exact size I am seeking after the nested PCR and electrophoresis. I extracted the DNA from the gel and sequenced it but the sequence I got is not matching to any pathogen on the blast!! Any one faced a similar problem? I also had a lot of overlapped waves after sequencing. I found latter that this primer attaches to Ehrlichia chaffeensis and Ehrlichia ruminintium. Thanks!
Since you seem to have the 16S sequence, why not going for MUCH BETTER LAMP PCR?
http://scholar.google.pl/scholar?hl=pl&q=LAMP+PCR&btnG=&lr=
Thank you Marcin Nowicki for the reply. Using the LAMP PCR will be a good idea but still can't identify the Ehrlichia strains from each other specially in case of co infection. The main problem I think is that these strains are genetically highly similar to each other that if there is a co infection between two or more strains of Ehrlichia the primer will attach to all strains so overlapping will happen and we'll never know which strains are coinfecting with which! Even I can't conclude that the bands which appeared after the nested PCR using the "species specific primer" for Mikurensis is Mikurensis because the sequencing did not confirm that!
There IS a solution.
IF there is even a single nucleotide difference, you can do HRM (high-resolution melting) of the LAMP PCR products, to discover differences. By comparing the HRM curves to single-template LAMP PCR HRM profiles, you can solve this ;)
Of course, 16S (ITS) is only only one of popular targets. You might want to check cox, Ypt1, B-tubulin, elicitin... (high copy genes) for suitable variants per species.
Thank you for the solution, I started figuring out how to do it.
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