Nature Reviews Microbiology | AOP, published online 3 June 2008; doi:10.1038/nrmicro1918 PERSpECtiVES related species; facilitate outbreak investiga- I N N OVAti ON tions and forensic analysis; facilitate rapid information sharing through a centralized Ibis T5000: a universal biosensor database; allow immediate testing of sam- ples, as well as high-throughput testing; and approach for microbiology have low per-sample analysis costs. The technology that we describe in this article was designed to meet these David J. Ecker, Rangarajan Sampath, Christian Massire, Lawrence B. Blyn, specifications. We have coupled broad Thomas A. Hall, Mark W. Eshoo and Steven A. Hofstadler amplification by PCR with electrospray ionization mass spectrometry (ESI–MS) Abstract | We describe a new technology, the Ibis T5000, for the identification of in a system that we call the Ibis T5000. pathogens in clinical and environmental samples. The Ibis T5000 couples nucleic The basic principle of operation is shown acid amplification to high-performance electrospray ionization mass spectrometry in FIG. 1. In brief, multiple pairs of prim- and base-composition analysis. The system enables the identification and ers are used to amplify carefully selected quantification of a broad set of pathogens, including all known bacteria, all major regions of the genome of the microorgan- ism of interest. Following amplification, groups of pathogenic fungi and the major families of viruses that cause disease in a fully automated ESI–MS analysis is humans and animals, along with the detection of virulence factors and antibiotic performed. The mass spectrometer effec- resistance markers. tively weighs the amplicons, or mixture of amplicons, with sufficient mass accuracy A walk into a clinical microbiology labora- health-care system. The CDC’s PulseNet that the composition of nucleotides (A, tory today can be like taking a step back programme (see Further information) for G, C and T) can be deduced for each in time: culture plates, incubators and the comparison of PFGE patterns perhaps amplicon present. The base composition microscopes dominate the ‘labscape’. It can presages the future of tracing and follow- is compared with a database of calculated take days to fully characterize the infectious ing accidental or intentional outbreaks of base compositions that is derived from the agents in a patient’s sample, beginning with infectious disease. However, PFGE is time sequences of known organisms to deter- culturing, followed by biochemical tests. consuming, requires a high level of skill mine the identities of any microorganisms Public-health laboratories also deal with the and the pattern results can vary between that are present. Thus, analysis using the detection and management of infectious laboratories. This demonstrates the primary Ibis T5000 provides detailed information diseases, but from the perspective of ben- hurdle that prevents rapid and reliable data which is analogous to that obtained using efiting society rather than the individual. sharing between clinical microbiology labo- a microarray or parallel DNA-sequencing Epidemiological investigations begin with ratories, public-health laboratories and bio- instrument. The Ibis T5000 mass spec- laborious methods, such as culturing, fol- defence agencies: the lack of a reproducible, trometer analyses each PCR reaction in lowed by pulse-field gel electrophoresis high-throughput, cost-effective technology less than 1 minute, using no consumable (PFGE) or sequencing. Government that provides digital signatures to detect and products, and is completely automated. We biodefence and homeland-security agencies identify microorganisms. have developed assays for use with the Ibis have a mandate to detect infectious disease The development of this type of technol- T5000 that enable the simultaneous iden- agents to protect against, or respond to, ogy is a daunting proposition. According to tification and quantification of all known acts of bioterrorism. The US government, literature reports, more than 1,400 species bacteria, all major groups of pathogenic for example, has built a large surveillance of microorganism are known to cause fungi and the major families of viruses infrastructure to monitor the environment disease in humans1. Moreover, each of that cause disease in humans. Here, we in major cities for the presence of biologi- these species can have hundreds of strain or describe how this technology can be used cal weapons that might be released in an genotypic variations with different proper- for pathogen detection for the benefit of aerosol attack. In these cities, individuals ties of virulence, transmissibility or drug individuals and society. with acute respiratory distress or fevers are resistance. The ideal diagnostic technol- treated in hospital emergency departments ogy to serve individuals, broad public- The Ibis T5000 universal biosensor and doctors’ offices. If an individual was health interests and biodefence should The mass spectrometer component of the infected with a microorganism of bioterror- provide universal pathogen-identification Ibis T5000 enables more information to ism significance, it would not be discovered capability; identify all organisms that are be extracted from PCR reactions than can be through the government’s efforts, as the present in a quantitative manner; identify obtained with standard individual probes. government’s environmental surveillance emerging, previously uncharacterized This enriched information extraction programme is not integrated into the organisms and determine the most closely occurs in two dimensions simultaneously. nature reViews | MICROBIOLOGY adVance OnLine puBLicatiOn | 1 © 2008 Nature Publishing Group PERS p ECTIVES 9 Conserved Variable Conserved species, and primers for bacteria can be as RNase P, and housekeeping proteins . designed that encompass the entire bacte- For this strategy, it is essential that the rial domain of life. Second, a large amount broad-range primers flank regions of vari- Primer design of information is obtained from each indi- ability such that the base composition of vidual amplicon by mass spectrometry. The the resulting amplicon is information rich. mass spectrometer weighs each amplicon It would be ideal if an amplicon from a with sufficient accuracy that the composi- single pair of broad-range primers would tion of nucleotides (As, Gs, Cs and Ts) can provide sufficient information to identify PCR be unambiguously determined. Although all bacteria and determine the species but, not as information rich as the sequence because some bacterial species have the (the linking order is not determined using same base composition in conserved primer ESI–MS), for many diagnostic purposes, regions, a number of amplicons from dif- the nucleotide composition of a nucleic ferent regions of the microbial genome acid can have the same practical value. must be analysed. Primer choices are made For example, when a small set of primers based on their ability to provide maximal ESI–MS is strategically chosen, approximately six parsing using base composition as a metric. PCR reactions can yield sufficient infor- Microorganisms are distinguished using the mation to identify the bacteria that are aggregate information from the base com- present to the species level2. For viruses, position of a number of amplicons; we refer primers can be designed to encompass to this as triangulation. The resolving power broad genera, such as Alphaviruses3 or of base-composition analysis is compared Mastadenoviruses4, or even whole virus with that of sequencing in BOX 1. families, such as the Orthomyxoviridae5 Analysis of a sample using multiple broad 6 Base-composition or Coronaviridae . When primers are PCR reactions has several other important assignment designed to amplify all known members benefits. The first relates to coverage of within a target group, previously uncharac- diverse organisms. Broad-range primers terized members are also detected. This is designed to amplify DNA from all bacteria, a crucial advantage of the Ibis T5000 tech- even those targeted to ribosomal DNA, do nology relative to probe-based molecular not match all organisms perfectly and a methods, for which anticipation of the certain number of mismatches between the target nucleic acid sequence is required to PCR primers and some genomes is inevita- design the probe. ble. This was considered in the development Figure 1 | Flow scheme for Ibis T5000 analysis. of PCR conditions for the Ibis T5000, and Like any other methodology that uses PCR, Bacterial surveillance the conditions are permissive in the first few Nature Reviews | Microbiology the nucleic acids must first be purified from the Detection and identification of multiple cycles to ensure hybridization of a partially clinical or environmental sample. Target sites bacterial species in a clinical sample, such mismatched oligonucleotide primer to on microbial genomes that are common across as blood, cerebrospinal fluid or sputum, is the target. Great care is taken to match the broad groups of microorganisms and that flank challenging. The conventional molecular 3′ ends of the primers to the targets; this regions of high information content are approach is to design specific PCR primers region is the most sensitive to mispairings selected for primer design. Broad-range PCR is conducted and, following an automated desalting that target specific pathogens. However, during PCR initiation. However, priming step, amplified nucleic acids