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.

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

The Problem: Bacillus anthracis

Worried Well?

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

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

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

Partnership: Biology & Engineering

DNAProbes

DNAProbes

Microwaved DNAfrom bacteria

Microwaving Spores?

BEFORE

AFTER AFTER

AFTER

E.g. MW C. difficile spore

B. anthracis spore biology

Bacillus Screening

(Adapted from Otter, A et al., Submitted)

• Representative panel of 56 isolates• Incl. DNA from virulent B. anthracis for

pXO2

gDNA Extraction

56 strains from library

gDNA standardised to 0.08ug/ml

5 SECONDSX microwave power

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)

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

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.

Detection of pX01 (5 mins)

Negative detection of pX01 (5 mins)

Detection of pX02

• Identified Probes for pXO2 detection via bioinformatics

• Screening against our representative panel

• Incl. virulent B. anthracis

• Aim to highlight False Positives

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

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

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

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

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