Download Phage Based Diagnostic Systems

LECTURE 17:
Phage Based Diagnostic Systems
Viro102:
Bacteriophages & Phage Therapy
3 Credit hours
Atta-ur-Rahman School of Applied Biosciences (ASAB)
PHAST SWAB, A Recombinant Reporter
Phage
• The Phast Swab
–Reporter bacteriophage,
– Immuno-magnetic separation,
– Bacterial enrichment in one easy to
use device
PHAST SWAB, A Recombinant Reporter
Phage
• Reporter
bacteriophages
represent a novel and
sensitive alternative to
conventional methods
for the detection of
bacteria
PHAST SWAB, A Recombinant
Reporter Phage
• The Phast Swab is a
simple to use device.
• The bottom of the
device contains
bacterial growth media
and specific
immunomagnetic (IMS)
beads.
• The swab is removed, the surface to be tested is swabbed, and the swab
is returned to the device, followed by an 8 hour enrichment.
• Following enrichment, the IMS beads (with target bacteria attached) are
concentrated, and the growth media is removed.
• Following a wash step, the reporter phage is mixed with the target
bacteria (this is accomplished directly in the device) and the Phast Swab
is incubated at 37oC for 1.5 hours.
• Finally, the cap of the Phast Swab is broken, releasing the betagalactosidase substrate into the bottom of the device, where it reacts
with any beta-galactosidase present.
• A positive test is indicated by the development of a red color, while in a
negative test, the color remains yellow
PHAST SWAB, A Recombinant
Reporter Phage
Phage Mediated Bioluminescent
ATP Assay
Phage Mediated Bioluminescent ATP
Assay
• What is the ATP bioluminescent assay?
• It is a method of accurately determining levels
of microbial ATP , and thus, the number of
bacteria in each sample
How it is carried out ?
Cell lysis
Release of ATP in
media
Detection of ATP through
firefly luciferase
Phage Mediated Bioluminescent ATP
Assay
• These assays are based on the fact that there
is a linear relationship between the number of
photons produced by firefly luciferase and the
number of ATP molecules hydrolyzed
• and the fact that the amount of ATP per
bacterial cell in a given growth condition is
quite constant (approximately 10^-15 g per
cell)
Limitations
Sensitivity
Specificity
Increasing Sensitivity
• Cellular Adenylate kinase is caused to be
released along with ATP
• It is a phosphotransferase enzyme that
catalyzes the inter-conversion of adenine
nucleotides:
• 2 ADP
ATP + AMP
Effect of AK on sample
• Hence instead of using the amount of ATP as a
direct parameter, it is used indirectly by
employing the direct proportionality of AK
activity with the bioluminescent signal and,
ultimately, to the number of cells in the
sample.
Effect of AK on sample
• As AK deals not only with ATP but ADP as
well, it leads to an increased
bioluminescent signal
• Therefore, theoretically, it is possible to
detect even a single cell in a certain sample
Where do phages come in?
• Phages are used to solve the specificity
issue
• Specificity is enhanced by using phages to
lyse target cells, owing to their specific and
efficient attachment to host bacterium and
its subsequent lysis.
• While diagnosing a certain bacteria in a
sample, we use a phage with known
specificity for that bacteria
Adding the two together…
Using phages and adenylate kinase together
in a bioluminescent assay gives us a
bioluminescent signal that is:
1. Enhanced due to adenylate kinase
2. Specific due to bacteriophages
Comparison with other diagnostic
techniques
• Salmonella can be detected within 2 hours
using this phage mediated technique,
opposed to the previous 5 day long method
• Also, fewer then 104 cfu/ml of E. coli can be
detected in less then 1 h
Specimen processing
Decant supernatant.
Suspend in 1ml
Response Medium
Plus
Sputum or blood
collection
Add
0.5ml test
suspension
Decontaminate
by NaOH method.
RIF0.5ml
Response
Medium Plus
Suspend pellet in 1.5ml
Response Medium Plus.
Centrifuge at minimum
of 2000g for 20 mins
Incubate both vessels
for 18-24 hours.
(commence with
RIF+
0.5ml
Response
Medium Plus
ThankYou !