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 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

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

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

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)

Explain proportionality Arbitrary Light Units

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

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

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 Still one unsolved problem: specificity

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

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 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 18-24 hours. (commence with

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