title: 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.
authors: Ibis Biosciences, Abbott, Carlsbad, California, United States of America. rangarajan.sampath@abbott.com
authors: Rangarajan
authors: R
authors: Sampath
authors: Niveen
authors: N
authors: Mulholland
authors: Lawrence B
authors: LB
authors: Blyn
authors: Christian
authors: C
authors: Massire
authors: Chris A
authors: CA
authors: Whitehouse
authors: Nicole
authors: N
authors: Waybright
authors: Courtney
authors: C
authors: Harter
authors: Joseph
authors: J
authors: Bogan
authors: Mary Sue
authors: MS
authors: Miranda
authors: David
authors: D
authors: Smith
authors: Carson
authors: C
authors: Baldwin
authors: Mark
authors: M
authors: Wolcott
authors: David
authors: D
authors: Norwood
authors: Rachael
authors: R
authors: Kreft
authors: Mark
authors: M
authors: Frinder
authors: Robert
authors: R
authors: Lovari
authors: Irene
authors: I
authors: Yasuda
authors: Heather
authors: H
authors: Matthews
authors: Donna
authors: D
authors: Toleno
authors: Roberta
authors: R
authors: Housley
authors: David
authors: D
authors: Duncan
authors: Feng
authors: F
authors: Li
authors: Robin
authors: R
authors: Warren
authors: Mark W
authors: MW
authors: Eshoo
authors: Thomas A
authors: TA
authors: Hall
authors: Steven A
authors: SA
authors: Hofstadler
authors: David J
authors: DJ
authors: Ecker
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D001681:Biological Assay; D054045:Biological Warfare Agents; D016000:Cluster Analysis; D017931:DNA Primers / Q000378:metabolism; D005188:False Negative Reactions; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000379:methods*; D058028:Research Report; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013223:Statistics as Topic; D014780:Viruses / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D054045:Biological Warfare Agents; D017931:DNA Primers