Maldi-TOF –v- PCR Probes in the Evaluation of Gastrointestinal Microflora
Written by 2016 Phoebe Wynne-Lewis, BHSc, Dip Nat Med, Dip Herb Med – FxMed Technical Support
Recent research into the gastrointestinal microbiome has confirmed the microbial inhabitants of the gastrointestinal tract, and their vast scope of metabolic activities, are at the very core of health and many disease processes. Microbiology testing should be optimized to address the abundance of all bacterial species present in a stool specimen.
This blog aims to summarize and weigh up the two current testing methodologies used to study gastrointestinal flora.
Proteomic Analysis (MaLDI-TOF)
This method is based on the culture method which is considered the industry gold standard as it enables identification of living metabolically active micro-organisms.
MaLDI-TOF technology identifies the micro-organisms by Proteomic Analysis which identifies the proteins in cells, tissues or organisms. Microorganisms have unique ribosomal protein ‘fingerprints’.
MaLDI-TOF stands for: Matrix- assisted Laser Desorption/Ionization Time of Flight Mass Spectroscopy
- MaLDI-TOF allows identification of over 7,000 micro-organisms! Traditional culture methods identify around 400.
- The stool sample is collected into a preservative solution called ‘Clary Blair Transport Medium’ to prevent continued growth of flora whilst keeping the micro-organisms alive. The medium is buffered to prevent shifts in pH during transport, and contains a reducing agent designed to markedly decrease oxygen levels to favour the facultative and strict anaerobic species known to inhabit the GI tract. When properly collected and shipped, beneficial and pathogenic species may be successfully recovered quantitatively from the transport media for up to 14 days. showing proof of this concept – figure 1
- MaLDI-TOF has been used to study a host of macromolecules from a wide variety of biological matrices, and similar methods of detection have been used by NASA on the Mars Curiosity Rover in an effort to determine if the planet had been hospitable for microbial inhabitance.
- The MaLDI-TOF approach has an impressive capability to differentiate closely related species from a genus such as Bacillus.
- The results of an international, multicentre trial to evaluate the reproducibility of a commercially available MaLDI-TOF MS platform were presented at the Congress of Clinical Microbiology and Infectious Diseases in 2009. Sixty blind-coded bacterial specimens were sent to eight different international laboratories. A concordance rate of 98.8% was achieved among labs for the 480 specimens. Not only were the samples correctly identified across laboratories, but the reliability of identification was also very high as 97.3% of all log score values were > 2.
- Considering accuracy, inter laboratory reproducibility, cost and the rapidity of identification the proteonomic approach (MALDI-TOF MS) is best suited for routine comprehensive stool analysis.
The 5 steps in the MaLDI-TOF MS identification process:
(1) Ionization of proteins from a pure isolate (with a lazer)
(2) Separation of the vaporized, ionized proteins that have different masses (through the mass spectrometer)
(3) Detection of the number of the different ionized proteins
(4) Collection of the data
(5) Comparison for identification to a huge data base