tina joshi presentation on "development of a real time microwave-enhanced rapid (~5 minutes)...
TRANSCRIPT
![Page 1: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/1.jpg)
Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay
for Bacillus anthracis
Dr. Lovleen Tina Joshi*, Dr. Jonny Lees†, Prof. Adrian Porch†, R. Probert≠, Dr. T. Connor≠, Prof. Les Baillie*
*School of Pharmacy & Pharmaceutical Sciences, †School of Engineering, ≠School of Biosciences
Cardiff University
The Biology of Anthrax Conference 2016, Sirata Resort, St Pete Beach, Fl.
![Page 2: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/2.jpg)
BIOLOGY ENGINEERING
Prof. Les Baillie (PI)
Prof. Adrian Porch (CI)
Dr. Jonathan Lees (CI)Dr. L.T. Joshi
PDRA/ Project manager
Hayder Hamzah
DmitryMalyshev
Microwave BioDetection Research Group
Evans Ahortor
![Page 3: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/3.jpg)
The Problem: Bacillus anthracis
![Page 4: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/4.jpg)
![Page 5: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/5.jpg)
Worried Well?
![Page 6: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/6.jpg)
Current Detection Methods
• Rapid Systems:• Direct Microscopy (> 10 minutes)• Antibody Based
• Concerns of specificity and sensitivity• Quality control concerns• Cross reactivity?
• Relatively Rapid Systems:• Antibody and DNA based • >1 hour
• Traditional Methods:• Culture (> 1 Day)
• Requires BSL-3 Capabilities• Time consuming• Most sensitive as determined by
Amerithrax FBI Investigation
![Page 7: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/7.jpg)
What do we want to detect and why?
The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteriaRead et al., 2003. Nature 423, 81-86(1 May 2003)
• Genomic DNA target• Plasmids:
• pX01- Tripartite toxin- lethal factor (LF Toxin), PA and EF• pX02* - Capsule (CPS) Highly virulent strains
![Page 8: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/8.jpg)
Bioinformatic Screen “Big Data”
• Multi step analysis using published genomes- assess high homology, low or none
• Reference genome (B. anthracis) split into 60 bp fragments in silico
• 194 Bacillus genomes & 2862 plasmid sequences identified
• Fragments subjected to BLAST & screened
• Purpose: Identify sequence reads with high similarity in only B. anthracis samples
• Best hits (based upon highest sequence homology amongst B. anthracis and lowest homology to samples from outside of B. anthracis) were quantified
• Probes were designed from these anthracis- specific regions
![Page 9: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/9.jpg)
Partnership: Biology & Engineering
DNAProbes
DNAProbes
Microwaved DNAfrom bacteria
![Page 10: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/10.jpg)
Microwaving Spores?
BEFORE
AFTER AFTER
AFTER
E.g. MW C. difficile spore
B. anthracis spore biology
![Page 11: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/11.jpg)
Bacillus Screening
(Adapted from Otter, A et al., Submitted)
• Representative panel of 56 isolates• Incl. DNA from virulent B. anthracis for
pXO2
![Page 12: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/12.jpg)
gDNA Extraction
56 strains from library
gDNA standardised to 0.08ug/ml
5 SECONDSX microwave power
![Page 13: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/13.jpg)
Cardiff University In-House B. anthracis POCD (MKI)
HRP
Captured DNA complex HRP
2H2O2
2OPD DAP
2H2O + O2Streptavidin coated dot on Silver ink + Acetate Sensor Combs (Vantix Ltd)
HRP
MicrowavedC. difficile Target(released DNA)( ~43 nt)
HRP ReporterDNA Probe (22 nt)
Biotin
BiotinylatedAnchorDNA Probe (17 nt)
5 ntgap
Streptavidin coated dot
Joshi et al., 2016. Microwave-enhanced disruption and rapid DNA detection of Clostridium difficile spores (Submitted to Biosensors and Bioelectronics)
![Page 14: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/14.jpg)
Preliminary In house B. anthracis POCD (5 mins)
Detection of B. anthracis spores (10 mins)
-600
-400
-200
0
200
400
600
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 100
103
Elec
tric
al Si
gnal
gene
rate
d in
mill
iVol
ts
Time (seconds)
Average Signal B. anthracis Std microwaved (1+4)Average Signal B. anthracis Sterne Microwaved (1+4)Streptavidin Control
Water control
MW B. anthracis Sterne pXO1+ positive control
Water control
MW B. anthracis Sterne pXO1- negative control
-100 mV
![Page 15: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/15.jpg)
Detection of genomic target (5 mins)
B. anthracis ΔgerH; B. anthracis Sterne and B. cereus ATCC 6464 (negative
control). Results demonstrate that the probes only detect B. anthracis.
![Page 16: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/16.jpg)
Detection of pX01 (5 mins)
![Page 17: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/17.jpg)
Negative detection of pX01 (5 mins)
![Page 18: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/18.jpg)
Detection of pX02
• Identified Probes for pXO2 detection via bioinformatics
• Screening against our representative panel
• Incl. virulent B. anthracis
• Aim to highlight False Positives
![Page 19: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/19.jpg)
Conclusions
• Identification of conserved gene targets• Genome & plasmids
• Results indicate probes are specific to B. anthracis ONLY• Finishing final pXO2 lab testing• Forward to Field tests
• Establish LoD in real samples
![Page 20: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/20.jpg)
The Final Vision...
Gold contact points
Microwaving area
Sample enters microfluidic capillary
Gold Sensor where DNA
detection occurs
Gold nanolayerdeposited on clear glass
DNA transistors
![Page 21: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/21.jpg)
A prototype detector that works in 5 minutes
• Rapid, accurate detection
• DNA detected in < 5 minutes WITHOUT prior purification like PCR
• Diagnostics need to be easy to use, reliable and compact
• Can be operated with minimal training
• Simple Yes/ No answers
• Low logistical footprint
• Can be used to detect bacteria in hospitals, rapid triage tool to weed
out the worried well
• Environmental survey tool to detect environment contamination and
identify no/ go area and areas that need to be decontaminated
![Page 22: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/22.jpg)
Conclusions: Impact
• Our technology has the potentialto underpin a wide range ofapplications beyond healthcareand security. Generate $60-110million /year in global sales.
ECONOMY
• Will save lives• Platform can be adapted to detect
other clinically relevant pathogens • Reduce use of antibiotics• Prevent infections spreading
• Facilitate the rapid detectionof biological agents such asAnthrax by first responders
• A simple to use detector, working in 5 minutes would improve diagnosis and patient satisfaction
• Change the diagnostics landscape, increasing development of new ideas and research
SOCIETYHEALTH SECURITY
![Page 23: Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis"](https://reader035.vdocuments.site/reader035/viewer/2022062902/58edb5851a28ab82098b467d/html5/thumbnails/23.jpg)
Acknowledgements
• Cardiff School of Pharmacy & Pharmaceutical Sciences:• Colleagues in Lab 1.11
• Prof. Les Baillie • Cardiff School of Biosciences:
• Dr. Tom Connor for Bioinformatics Screening• Cardiff School of Engineering:
• Prof. Adrian Porch, Dr. Jonny Lees • Hayder Hamzah
• Dr. Emmanuel Brousseau