PCR-electrospray ionization mass spectrometry: the potential to change infectious disease diagnostics in clinical and public health laboratories

Abstract

During the past 20 years, microbial detection methods that are genetically based, such as real-time PCR and peptide nucleic acid fluorescent hybridization, coexisted with traditional microbiological methods and were typically based on the identification of individual genetic targets. For these methods to be successful, a potential cause of infection must be suspected. More recently, multiplex PCR and multiplex RT-PCR were used to enable more broad-range testing based on panels of suspected pathogens. PCR-electrospray ionization mass spectrometry (PCR-ESI/MS) has emerged as a technology that is capable of identifying nearly all known human pathogens either from microbial isolates or directly from clinical specimens. Assay primers are strategically designed to target one or more of the broad pathogen categories: bacterial, mycobacterial, fungal, or viral. With broad-range amplification followed by detection of mixed amplicons, the method can identify genetic evidence of known and unknown pathogens. This unique approach supports a higher form of inquiry, asking the following question: What is the genetic evidence of known or unknown pathogens in the patient sample? This approach has advantages over traditional assays that commonly target the presence or absence of one or more pathogens with known genetic composition. This review considers the breadth of the published literature and explores the possibilities, advantages, and limitations for implementation of PCR-ESI/MS in diagnostic laboratories.

Figure 1

A: The Ibis T5000 Biosensor System is the PCR-ESI/MS prototype. B: The Abbott PLEX-ID system is the current instrument.

Figure 2

Conserved sequences serve as priming sites on which broad-range primers amplify sequences of various sizes and compositions, because of combinations of conserved regions that flank intercalated regions of sequence diversity, found in the space between common regions.

Figure 3

Representative mass spectra from PCR-ESI/MS. Data resulting from ESI TOF MS of each microtiter well containing one primer pair and the target DNA. A: The 96-well view of spectra collected. B: Two charge-state distributions are seen in products produced with primer 346 for each forward and reverse primer, indicating the presence of two microorganisms in this sample. Blue, Bacteroides thetaiotaomicron; green, S. aureus. C: Deconvolution of collected mass spectra, resulting in a mol. wt. for the forward and reverse strand. The resulting base compositions are as follows: [30A-27G-23C-19T] (top panel) and [27A-30G-21C-21T] (bottom panel).

Figure 4

The ESI/MS generates amplicon base composition values from the intact mol. wt. measured by MS for several genetic regions and compare results to the Ibis microbial sequence database. Composition values will vary by microorganism.