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.

Broad-Spectrum Biosensor Capable of Detecting and Identifying Diverse Bacterial and Candida Species in Blood

“…PCR amplifications were performed using previously described PCR reagents and cycling conditions (15,16). PCR products were desalted by binding to magnetic microparticles, washing with solutions containing volatile salts and organic solvents, and then eluting with a high-pH buffer solution containing 35% (vol/vol) methanol and 25 mM piperidine-imidazole, as previously described (17).…”

“…Desalted PCR products were injected into an electrospray ionization-time of flight mass spectrometer (ESI-TOF-MS) (17). Peptide-based mass standards were used to bracket the expected mass-to-charge range and minimize TOF-associated variability.…”

Emerging Technologies for the Clinical Microbiology Laboratory

“…ESI-MS has been applied to the identification of bacterial pathogens obtained from a variety of matrices, including food, cell cultures, environmental samples, and positive blood culture broths (226)(227)(228)(229)(230). A multiplex PCR panel coupled with ESI-MS was evaluated for the identification of food-borne pathogens and demonstrated 99% to 100% sensitivity for Salmonella and E. coli isolates, including accurate serotyping for 30% of Salmonella isolates (229); however, identification of Shigella isolates was variable, ranging from 100% (S. sonnei) to 42.1% (S. dysenteriae).…”

“…Direct analysis of food samples containing enteric pathogens was also variable, with a sensitivity of less than 50% for tomatoes and chili powder but 100% for cheese and fish (229). ESI-MS has been also utilized to identify and differentiate species within 14 "biothreat clusters," which include biothreat agents and closely related species (e.g., Francisella tularensis, F. novicida, and F. philomiragia) (226,230). Based on a panel of 36 primer pairs and ESI-MS, the assay demonstrated analytic sensitivity as low as 40 genome equivalents and was 100% specific even when specimens contained an excess of a closely related, nonpathogenic organism (230).…”

“…ESI-MS has been also utilized to identify and differentiate species within 14 "biothreat clusters," which include biothreat agents and closely related species (e.g., Francisella tularensis, F. novicida, and F. philomiragia) (226,230). Based on a panel of 36 primer pairs and ESI-MS, the assay demonstrated analytic sensitivity as low as 40 genome equivalents and was 100% specific even when specimens contained an excess of a closely related, nonpathogenic organism (230). This methods was also evaluated using simulated specimens, including tissue, food, and environmental samples, and demonstrated similar performance (226).…”