Microorganism identification techniques are evolving, increasingly favoring PCR-based methods. Indeed, PCR amplification coupled with Sanger sequencing enables the identification of bacteria and yeast. This is achieved by referencing the sequences in the 16S and ITS databases using BlastTM on NCBI. This approach helps identify bacteria or yeast that may produce ambiguous results in traditional galleries.
Please note that for certain genera of bacteria, the sequence may resemble that of two very similar species.
Technique
Identification is carried out by PCR using genomic DNA, colony, or culture broth. The primers are specific to the 16S and ITS regions.
16S-Bacteria
16S-27F AGAGTTTGATCCTGGCTCAG
16S-1541R AAGGAGGTGATCCAGCCGCA
Amplicon: About 1500 pb
ITS-Yeast
ITS1 TCCGTAGGTGAACCTGCGG
ITS4 TCCTCCGCTTATTGATATGC
Amplicon: Variable depending on the species, ranging from 600 to 900 bp.
PCRs are purified, quantified, diluted, and sequenced using the Sanger method. The results are provided in the form of chromatograms and DNA sequences. With this sequence, you will be able to perform a BLAST and compare your sequence with those deposited in the database. This will allow you to determine the percentage of homology with certain species of bacteria or yeast, thereby confirming the identification.
Our current processing time is approximately 1 week. Typically, we process samples on Wednesday afternoon, and the results are available on Monday morning.