Comprehensive biothreat cluster identification by PCR/electrospray-ionization mass spectrometry

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.

Figure 1. Biothreat clusters of interest.

Ten bacterial and four viral clusters identified in the biothreat assay are shown. In each cluster the key biothreat agent and its near neighbors are indicated. The HHS/USDA select agent and NIAID A, B, C pathogen lists are reflected by symbols shown in the legend. Attenuated or live vaccine strains of some of these organisms are, however, excluded from the select agent list (http://www.selectagents.gov/Select Agents and Toxins Exclusions.html). B. anthracis and Y. pestis plasmid markers are explicitly annotated. Organisms with names given within brackets cannot be distinguished from each other within this assay. Primer pairs used for the detection of each biocluster are indicated.

Figure 2. Linearity of response of the biothreat assay.

B. anthracis DNA was spiked into AE buffer (Clean) or an extract from an environmental air filter (Dirty) at concentrations ranging from 0–1000 GE/well. The reported response from the PLEX-ID system was linear over the entire concentration range tested in both cases.