Like our AccuGENX-ID® methods for identification of bacteria and fungi using the 16S and ITS2 genes, our AccuGENX-ST® sequence typing uses standard molecular biological methods of DNA extraction, PCR amplification and DNA sequencing of protein coding target genes to characterize your isolates to the strain level. The resulting DNA sequences, or sequence types, are compared to others in a database and the relationships displayed in a phylogenetic tree. Charles River will perform comparisons for any samples or sets of samples upon request, all at no additional charge to you.
Tracking sources of contamination
If a sterility failure occurs, customers can send samples to us, and we can assist in determining the root cause. We will first identify the isolates by 16S rDNA sequencing. If the 16S sequence is different, you can be sure that the isolates are different strains. Further characterization is necessary for those isolates that have identical 16S sequences and can be done using SLST or MLST. This technology is designed to deliver the most informative data and have the ability to obtain an unambiguous result from all strains within a species.
Our sequence-based strain typing can help you map your microbial environment. We can create a library database of sequences from the microorganisms found in your manufacturing environment. When a contaminant is encountered, you can quickly pinpoint where you have seen that particular strain before. This greatly reduces the time and cost associated with identifying the source of contamination.
Methods for sequence-based strain typing
Microbial identification using the rRNA regions is a robust technology. There are times, however, when additional information is needed and it is essential to be able to differentiate organisms below the species level. Increased discrimination at the strain level can be achieved by sequencing, analyzing and comparing highly variable loci (regions) in the organism’s genome. MLST and SLST analyze essential protein coding genes, or housekeeping genes, that encode for proteins necessary for the normal cellular functions of the bacterium all of which contain more variability in their sequences.
After extracting DNA from the sample, which can be viable or non-viable, the first step in this process is an accurate ID to the species level. This must be performed before starting MLST or SLST to determine appropriate targets and primer sets as the target genes will not be the same for each species being characterized. The target genes are PCR amplified using the gene-specific primers and subjected to comparative DNA sequence analysis. All the sequences from each gene target are aligned and compared to the other organisms’ sequence data and the level of divergence or conservation between the organisms is calculated and displayed with a phylogenetic tree. We will interpret the data for you and state whether the isolates are indistinguishable by the sequencing of the particular gene targets or whether the isolates are different sequence types.