Abstract
Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.
Highlights
Technology for detecting biothreat agents requires accurate identification of a broad array of bacterial and viral organisms that can cause severe disease and/or death, whether they occur as a result of a biological attack or from a natural source in the environment
These organisms make up the majority of the National Institute of Allergy and Infectious Diseases (NIAID) Category A, B, and C priority pathogens and Health and Human Services (HHS)/United States Department of Agriculture (USDA) select agents
The biothreat assay described here identifies ten bacterial and four viral biothreat clusters included in the NIAID priority pathogen (Category A: seven agents, Category B: 18; Category C: three) and HHS/USDA select agent (18 agents) lists
Summary
Technology for detecting biothreat agents requires accurate identification of a broad array of bacterial and viral organisms that can cause severe disease and/or death, whether they occur as a result of a biological attack or from a natural source in the environment. The National Institute of Allergy and Infectious Diseases (NIAID) has compiled a list of priority pathogens for biodefense (http://www.niaid.nih.gov) and several of these are defined as select agents (http://www.selectagents.gov/) by various agencies such as Health and Human Services (HHS) and the United States Department of Agriculture (USDA) (some of the vaccine and live attenuated strains are, excluded from the select agents list: http://www.selectagents.gov/Select%20 Agents%20and%20Toxins%20Exclusions.html). These bioagents are often virtually indistinguishable from a group of phylogenetically related species or subspecies often referred to as ‘‘near neighbors’’ [1]. Effective biosensor technology must be capable of identifying a broad array of biothreat agents and distinguishing these threats from their near neighbors unambiguously
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