3 research outputs found
Detection of Yersinia pestis in Environmental and Food Samples by Intact Cell Immunocapture and Liquid ChromatographyâTandem Mass Spectrometry
Yersinia pestis is the causative
agent of bubonic and pneumonic plague, an acute and often fatal disease
in humans. In addition to the risk of natural exposure to plague,
there is also the threat of a bioterrorist act, leading to the deliberate
spread of the bacteria in the environment or food. We report here
an immuno-liquid chromatographyâtandem mass spectrometry (immuno-LCâMS/MS)
method for the direct (i.e., without prior culture), sensitive, and
specific detection of Y. pestis in
such complex samples. In the first step, a bottom-up proteomics approach
highlighted three relevant protein markers encoded by the Y. pestis-specific plasmids pFra (murine toxin) and
pPla (plasminogen activator and pesticin). Suitable proteotypic peptides
were thoroughly selected to monitor the three protein markers by targeted
MS using the selected reaction monitoring (SRM) mode. Immunocapture
conditions were optimized for the isolation and concentration of intact
bacterial cells from complex samples. The immuno-LCâSRM assay
has a limit of detection of 2 Ă 10<sup>4</sup> CFU/mL in milk
or tap water, which compares well with those of state-of-the-art immunoassays.
Moreover, we report the first direct detection of Y.
pestis in soil, which could be extremely useful in
confirming Y. pestis persistence in
the ground
Phage Amplification and Immunomagnetic Separation Combined with Targeted Mass Spectrometry for Sensitive Detection of Viable Bacteria in Complex Food Matrices
We
have developed and describe here for the first time a highly
sensitive method for the fast and unambiguous detection of viable <i>Escherichia coli</i> in food matrices. The new approach is based
on using label-free phages (T4), obligate parasites of bacteria, which
are attractive for pathogen detection because of their inherent natural
specificity and ease of use. A specific immunomagnetic separation
was used to capture the progeny phages produced. Subsequently, T4
phage markers were detected by liquid chromatography coupled to targeted
mass spectrometry. Combining the specificity of these three methodologies
is of great interest in developing an alternative to conventional
time-consuming culture-based technologies for the detection of viable
bacteria for industrial applications. First, optimization experiments
with phage T4 spiked in complex matrices (without a phage amplification
event) were performed and demonstrated specific, sensitive, and reproducible
phage capture and detection in complex matrices including LuriaâBertani
broth, orange juice, and skimmed milk. The method developed was then
applied to the detection of <i>E. coli</i> spiked in foodstuffs
(with a phage amplification event). After having evaluated the impact
of infection duration on assay sensitivity, we showed that our assay
specifically detects viable <i>E. coli</i> in milk at an
initial count of â„1 colony-forming unit (cfu)/mL after an 8-h
infection. This excellent detection limit makes our new approach an
alternative to PCR-based assays for rapid bacterial detection
Bacterial Detection Using Unlabeled Phage Amplification and Mass Spectrometry through Structural and Nonstructural Phage Markers
According to the World Health Organization,
food safety is an essential
public health priority. In this context, we report a relevant proof
of feasibility for the indirect specific detection of bacteria in
food samples using unlabeled phage amplification coupled to ESI mass
spectrometry analysis and illustrated with the model phage systems
T4 and SPP1. High-resolving power mass spectrometry analysis (including
bottom-up and top-down protein analysis) was used for the discovery
of specific markers of phage infection. Structural components of the
viral particle and nonstructural proteins encoded by the phage genome
were identified. Then, targeted detection of these markers was performed
on a triple quadrupole mass spectrometer operating in the selected
reaction monitoring mode. <i>E. coli</i> at 1 Ă 10<sup>5</sup>, 5 Ă 10<sup>5</sup>, and 1 Ă 10<sup>6</sup> CFU/mL
concentrations was successfully detected after only a 2 h infection
time by monitoring phage T4 structural markers in LuriaâBertani
broth, orange juice, and French bean stew (âcassouletâ)
matrices. Reproducible detection of nonstructural markers was also
demonstrated, particularly when a high titer of input phages was required
to achieve successful amplification. This strategy provides a highly
time-effective and sensitive assay for bacterial detection