Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing

Abstract

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy.

Rapid Detection of Bacillus anthracis Spores Using Immunomagnetic Separation and Amperometry

“…Overall, compared to reported conventional ELISAs for anthrax spore detection [4,22], PHAD has improved sensitivity, robustness and/or substantially reduced run times. Nucleic acid sequence (DNA or RNA), dipicolinic acid (DPA), and surface resonance (Raman spectroscopy)-based methods of anthrax spore detection all require sample processing procedures (often including sample enrichment) and equipment limiting the portability and practicality in the field [6,23,24,25]. …”

“…Current field-deployable methods of anthrax spore detection such as lateral-flow devices (LFDs) lack sensitivity and quantitative ability, and are subjectively interpreted [1,2,3]. More sensitive detection methods such as ELISA or PCR typically use time-consuming protocols and instruments not suitable for field use or require sample enrichment or clean-up prior to testing to avoid inhibition by environmental contaminants [4,5,6,7]. …”

Biological Warfare Agents and their Detection and Monitoring Techniques (Review Paper)

“…NGS platforms have become important components of an effective biodefense strategy because of their capability to detect and identify BWAs. NGS analysis has been utilised for detection of B. anthracis from soil and air samples and it could detect as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid 46 . NGS has also been used to detect strain-specific polymorphism in B. anthracis and Y. pestis 47 .…”

imGLAD: Accurate detection and quantification of target organisms in metagenomes

“…Detection limits vary depending on the sequencing effort and technology (e.g., read length), and the complexity of the microbial community sampled, and in most cases these parameters or their effects on the limit of detection remain inaccessible. Experiments with increasing amounts of target DNA added to environmental samples have been performed in the past to empirically establish detection limits [e.g., (1)]. However, a theoretical framework to establish limit of detection based on bioinformatics analysis of metagenomics is still lacking.…”