Hi, I am working the isolation and identification of environmental mycobacteria. For Identification of them, I used PCR and sequence of 16S rRNA and hsp65 genes. Of 16S rRNA and hsp65 genes, 473bp and 441bp amplified respectively. In the BLAST of results, I used the BioEdit, ChromasPro and DNALasergene softwares, but the results were not satisfactory and some species were not distinguished. Is the partial sequence of 16S rRNA and hsp65 genes can be identified mycobacteria species and subspecies????
With best regards,
M. Roshdi
Hi M. Roshdi, sequence of 16S rRNA and hsp65 genes are general used for microbial identification. Maybe you can used the pipeline on this site for identification.......... http://bioinfo.biotek.bppt.go.id/mident/
Sequencing parts of 16S rDNA and hsp65 is a good approach. However, your 16S fragment may be too small and even when combining both loci you may not be able to identify 100% of your isolates (see reference). In addition, sequencing of the ITS-region has been proposed by Harmsen et al (2003).
A comprehensive algorithm for molecular differentiation of Mycobacteria had been published many years ago by Dostal et al on the RIDOM website (however, it has been removed from the website - or at least I cannot find it anymore). For some species, amplification of IS-elements (IS 900, IS 902 or IS 2404) were proposed in addition to 16S rDNA sequencing (using the primes 16S-27f and 16S-907r as published by Lane in 1991).
http://www.sciencedirect.com/science/article/pii/S2212553114000314
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC280682/
Did you check quality of sequences? Since you used partial sequence of 16S rRNA, you have to obtain 100% chromatogram of sequence. It might be good if you use complete sequence of 16S rRNA. The other way is rpoB gene analysis.
Dear Oliver Nolte thank you for your response.
Best regards,
M. Roshdi
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