Description:
Researchers at TGen and Northern Arizona University have developed a multi-agent multi-locus amplicon sequencing protocol encompassing 79 targets aimed at detecting the presence or absence of five major biothreat agents: Burkholderia pseudomallei, Burkholderia mallei, Bacillus anthracis, Yersinia pestis and Francisella tularensis, as well as the presence and sequence of plasmids, virulence factors, antimicrobial resistance factors, and sequence variant loci for near-neighbor species differentiation with high sensitivity and specificity. The multiple amplicon sequencing system has a low limit of detection while still distinguishes distinguishing between a biothreat agent and its near-neighbor species using both amplification of positive/negative criteria and qualitative analysis of sequence within the amplicons. The multi-agent assay, consisting of two multiplex amplification reactions, was validated against a diverse subset of target agent and near neighbor panels that were previously used to validate assays targeting individual agents. The screening panel of target organism strains as well as near-neighbor strains allowed for differentiation with 100% sensitivity for all target agents and 91-100% specificity.
B. pseudomallei, B. mallei, B. anthracis, Y. pestis and F. tularensis are all Tier 1 select agents, posing a potentially severe threat to public health. Current surveillance methods used by civilian healthcare, government, and military institutions rely upon single locus PCR techniques that allow for only presence/absence of SA results, which has been known to lead to false positives, especially due to the complexity of environmental samples including huge numbers of microorganisms, many of which can be highly similar target pathogens. With the cost of sequencing decreasing and the ability to combine higher and higher numbers of samples on a single run, amplicon sequencing provides a cost effective alternative to individual PCR methods. Targeted amplicon sequencing utilizing a universal amplicon indexing scheme provides a superior alternative to the current single locus PCR systems and enables the detection of multiple biothreat agents across multiple samples with a single sequencing run, as well as the flexibility to adapt for detecting new biothreat agents and signatures.
