1. 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)

4. PHAST SWAB, A Recombinant Reporter Phage
The Phast Swab
Reporter bacteriophage,
Immuno-magnetic separation,
Bacterial enrichment in one easy to use device

5. PHAST SWAB, A Recombinant Reporter Phage
Reporter bacteriophages represent a novel and sensitive alternative to conventional methods for the detection of bacteria

6. 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.

7. 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 beta-galactosidase 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

8. PHAST SWAB, A Recombinant Reporter Phage

9. Phage Mediated Bioluminescent ATP Assay

10. 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

11. Detection of ATP through firefly luciferase
How it is carried out ?
Cell lysis
Release of ATP in media
Cell lysing agents
Explain firefly luciferase: uses ATP to generate liminescence (bioluminescent reaction)
Direct proportionality of ATP and luminescence generated
Detection of ATP through firefly luciferase

12. 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)

13. Explain proportionality
Arbitrary Light Units

14. Limitations Sensitivity Specificity
Sensitivity: at least 10^3 bacteria, practically 4 to 5
Specificity: do not know source of ATP, every cell in sample lysed, then how to apply this in diagnostics?
Only microbial load, not its composition

15. 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
However, ways exist to overcome these limitations

16. 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.

17. 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

19. 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
Still one unsolved problem: specificity

20. Adding the two together…
Using phages and adenylate kinase together in a bioluminescent assay gives us a bioluminescent signal that is:
Enhanced due to adenylate kinase
Specific due to bacteriophages

21. 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

23. Specimen processing Decant supernatant. Suspend in 1ml
Response Medium Plus
Specimen processing
Sputum or blood
collection
Add
0.5ml test
suspension
Decontaminate
by NaOH method.
RIF-
0.5ml Response
Medium Plus
RIF+
0.5ml Response
Medium Plus
Suspend pellet in 1.5ml
Response Medium Plus.
Centrifuge at minimum
of 2000g for 20 mins
Incubate both vessels
for hours.
(commence with

24. ThankYou !