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Published by Tim Sandle

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Published by: Tim Sandle on Jun 12, 2012
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Highly accurate strain analysis through sequence typing
 Dr. Christine Farrance, Senior Application Scientist, Accugenix Inc.In response to customer demands for more accurate and reliable techniques for strain leveldifferentiation of microorganisms, Accugenix is now offering cGMP compliant single- and multi-locussequence typing methods as an alternative to ribotyping. These methods represent a more accurate,reproducible and cost-effective approach to differentiate closely-related microorganisms.Tracking and trending of microorganisms and investigations of excursions in manufacturing settings areimportant parts of a comprehensive environmental monitoring program. Likewise, accurateconfirmation of identity of master cells banks, dietary ingredients and supplements is critical. Standardgenotypic sequence-based identification systems, utilizing regions of the ribosomal DNA (rDNA),increase the ability to accurately identify and track and trend microorganisms at the species level byusing the sequence rather than just the
name. However, some common contaminantsisolated from sterile and non-sterile manufacturing facilities cannot be resolved by this approach aloneand some cell banks, ingredients and supplements need a more in depth characterization. By combiningstandard genotypic identification methods with multi-locus sequence typing (MLST) or single-locussequence typing (SLST) it is possible to resolve some of the most difficult organisms to trend and track inmanufacturing industries.MLST and SLST are well-established, highly accurate sequence-based methods that are used todistinguish closely-related microorganisms to the strain level. Strain level differentiation is made byanalyzing protein coding or housekeeping genes that encode for proteins necessary for the normalcellular functions and which contain more sequence variability. Since more variation can be detected byanalyzing the differences in gene sequences, it results in a greater differentiation of subtypes in apopulation of organisms. The analysis of multiple loci provides additional variation to furtherdifferentiate between closely related strains. This genotypic technique is designed to deliver the mostinformative data and, as a result, is highly discriminatory.Since the foundation of M/SLST is built upon DNA sequencing results,which can be easily cataloged and referenced, these techniques are highlyreproducible, unambiguous and scalable as opposed to ribotyping.Additionally, databases can be created for historical trending and tracking.Given the reproducibility of M/SLST between experiments, and over time,these methods can be used more reliably to determine if isolatesrecovered from one area are the same or different as another isolate
atrait that allows for high-resolution trending and tracking projects.Confirmation of species identity for all isolates is first determined bystandard rDNA sequencing (16S or ITS2). Accurate identification isessential, as the target genes for sequence typing can be different foreach species. In general, SLST methods involve sequencing one gene thatis known to harbor variable DNA sequences, and with MLST multiple
2genes of moderate variability are sequenced. The gene sequences from each isolate are then alignedand compared in a phylogenetic tree to show the amount of conservation and divergence in the DNA of that gene region. The goal is to determine a gene or gene combinations that can give a high level of variability to differentiate to the strain level.As an illustration of this point, we present a case-study involving
Bacillus cereus,
an organism commonlyisolated from manufacturing environments. Additionally, while some strains of this organism areharmful to humans and can cause illness, others are beneficial and are used as probiotics. Isolates of 
are difficult to resolve or characterize with consistency by standard phenotypic or genotypicmethods, including 16S rDNA sequencing and ribotyping. However, MLST is capable of differentiatingisolates to the strain level, enabling reliable tracking and trending of this organism. The target genes,
, and
A, were amplified using specific primers and subjected to comparative DNAsequencing analysis. The sequences were aligned and compared to the
other organisms’ seque
nce data.The percent divergence between organisms was calculated and assembled into phylogenetic trees. Thedata from the three targets were used for strain level differentiation. Example trees indicating sequenceclusters or subtypes for 16S and
F are presented.Accugenix has always been committed to providing the most accurate and reliable species levelidentifications to our clients. Now, we are proud to include strain level characterization to our servicelist
We are the leader in innovation in the field of microbial identification and straintyping using genotypic methods. The development of MLST and SLST is just another example of howAccugenix continues to be the most innovative contract laboratory service provider by offering leading-edge cGMP compliant technologies as solutions for our clients.

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