Ibis T5000: a universal biosensor approach for microbiology

Abstract

We describe a new technology, the Ibis T5000, for the identification of pathogens in clinical and environmental samples. The Ibis T5000 couples nucleic acid amplification to high-performance electrospray ionization mass spectrometry and base-composition analysis. The system enables the identification and quantification of a broad set of pathogens, including all known bacteria, all major groups of pathogenic fungi and the major families of viruses that cause disease in humans and animals, along with the detection of virulence factors and antibiotic resistance markers.

Figure 1. Flow scheme for Ibis T5000 analysis.

Like any other methodology that uses PCR, the nucleic acids must first be purified from the clinical or environmental sample. Target sites on microbial genomes that are common across broad groups of microorganisms and that flank regions of high information content are selected for primer design. Broad-range PCR is conducted and, following an automated desalting step, amplified nucleic acids are electrosprayed into a time-of-flight (TOF) mass spectrometer and the spectral signal is processed to identify the masses of the amplicons that are present with sufficient mass accuracy to unambiguously calculate the nucleotide base compositions present in each amplicon. The information from all wells is used in a mathematical process that is known as triangulation to convert the base-composition data to a list of the organisms that are present and their relative and absolute quantities. ESI–MS, electrospray ionization mass spectrometry.

Figure 2. The bacterial domain of life depicted using 373 sequenced species and the coverage of 16 primer pairs.

The colour coding depicts the specificity of the broad-range primers that are used in bacterial identification. All the species shown are amplified by the six ribosomal-DNA-targeted primers (346, 347, 348, 349, 360 and 361); exceptions are shown boxed by a red dashed line, along with an indication of the actual subset of primer pairs that is predicted to yield amplicons. Other colours depict coverage of the division- or group-specific primers that target genes which encode conserved housekeeping genes. For clarity, diverse branches that are covered by ribosomal primer pairs only (the Aquificae, Thermotogae and Chloroflexi phyla) are not represented.

Figure 3. The spectrum of amplicons obtained from a sputum sample from a patient with cystic fibrosis using a primer pair that targets ribosomal DNA.

a | Shows a broad mass range that includes each strand of the internal calibration standard. b | Shows an expanded view of the spectral region that excludes the calibration standard. Each amplified product has two peaks that correspond to the two strands of the amplicon. The organisms that are assigned to each peak are based on the collective interpretation from eight broad-range primers.

Figure 4. Base-composition analysis of flaviviruses.

The base composition of a variable fragment of the RNA-dependent RNA polymerase gene flanked by conserved primer target sites is depicted in three dimensions for the flavivirus sequences in GenBank. The region depicted here corresponds to coordinates 9,050–9,150 of Dengue virus 1 (GenBank accession code DQ285559).