Tina Joshi presentation on "Development of a Real Time Microwave-Enhanced Rapid (~5 minutes) Detection Assay for Bacillus anthracis" at Biology of Anthrax, Tampa 2016

1. 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.

2. BIOLOGY ENGINEERING
Prof. Les Baillie
(PI)
Prof. Adrian
Porch (CI)
Dr. Jonathan
Lees (CI)Dr. L.T. Joshi
PDRA/ Project
manager
Hayder Hamzah
Dmitry
Malyshev
Microwave BioDetection Research
Group
Evans Ahortor

3. The Problem: Bacillus anthracis

4. • It has been predicted that
an attack against a city of
11.5 million inhabitants with
2.2 pounds of anthrax would
results in 1.5 million infections
(Wein et al., 2003)
• It has also been estimated
that the economic cost of an
aerosol attached against the
suburbs of a major city with
anthrax would cost
$26,000,000,000 per
100,000 persons exposed
(Kaufmann et al., 1997)
Human cost of an anthrax attack

5. Worried Well?
Triage
The process of determining the priority of patients' treatments based on severity of
their condition. This rations patient treatment efficiently when resources are insufficient
for all to be treated immediately. Wikipedia
Potential to overwhelm the system
Following the letter attacks in 2001, more than 10,000 people were offered antibiotics
to be taken for at least 60 days in conjunction with the licensed US anthrax vaccine

6. 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

7. What do we want to detect and why?
The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria
Read 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

8. 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

9. Partnership: Biology & Engineering
DNA
Probes
DNA
Probes
Microwaved DNA
from bacteria

10. Microwaving Spores?
BEFORE
AFTER AFTER
AFTER
E.g. MW C. difficile spore
B. anthracis spore biology

11. Bacillus Screening
(Adapted from Otter, A et al., Submitted)
• Representative panel of 56 isolates
• Incl. DNA from virulent B. anthracis for
pXO2

12. gDNA Extraction
56 strains from
library
gDNA standardised
to 0.08ug/ml
5 SECONDS
X microwave power

13. Cardiff University In-House B.
anthracis POCD (MKI)
HRP
Captured
DNA complex HRP
2H2O2
2OPD DAP
2H2O + O2
NH2 NH2 NH2
HRP N
Streptavidin
coated dot on
Silver ink +
Acetate
Sensor Combs
(Vantix Ltd)
HRP
Microwaved
C. difficile Target
(releasedDNA)
( ~43 nt)
HRPReporter
DNAProbe
(22 nt)
Biotin
Biotinylated
Anchor
DNAProbe
(17 nt)
5nt
gap
Streptavidin
coated dot
Joshi et al., 2016. Microwave-enhanced disruption and rapid DNA detection
of Clostridium difficile spores (Submitted to Biosensors and Bioelectronics)

14. Preliminary In house
B. anthracis POCD (5 mins)
Detectionof B. anthracis spores (10 mins)
-600
-400
-200
0
200
400
600
1
4
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46
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52
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82
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91
94
97
100
103
ElectricalSignalgeneratedinmilliVolts
Time (seconds)
Aver
anth
micro
Aver
anth
Micr
Strep
Wate
MW B. anthracis Sterne pXO1+ positive control
Water control
MW B. anthracis Sterne pXO1- negative control
-100 mV

15. 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.

16. Detection of pX01 (5 mins)

17. Negative detection of pX01 (5 mins)

18. Detection of pX02
• Identified Probes for pXO2 detection via bioinformatics
• Screening against our representative panel
• Incl. virulent B. anthracis
• Aim to highlight False Positives

19. 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

20. The Final Vision...
Gold contact
points
Microwaving
area
Sampleenters
microfluidic
capillary
Gold Sensor
whereDNA
detection occurs
Gold nanolayer
deposited on
clear glass
DNA transistors

21. 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

22. Conclusions: Impact
• Our technology has the potential
to underpin a wide range of
applications beyond healthcare
and security. Generate $60-110
million /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 detection
of biological agents such as
Anthrax 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

23. 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