Dianne Gleeson

The application of environmental DNA as a

biosecurity surveillance tool

What is eDNA?

• DNA that is left behind in the environment, sources

include water, soil, air, fecal etc

Methodology

eDNA Detection variables

Sensitivity depends upon:Volume of water, vs

Number of samples, ns

Number of aliquots, na

Proportion of template DNA per aliquot, ƒ

Developing quantitative framework

• Sensitivity depends upon:– θ, dispersion parameter

– λ, concentration of DNA

– ms , DNA molecules per water

sample

– mt, total molecules across all water

samples

– ma, no. molecules in an aliquot

– Xa, no. molecules amplified

– p, probability of successful

amplification of a target DNA

molecule

© Biosecurity SA

• Probability that no molecules will be

amplified in a single aliquot

P Xa = 0 | l > 0( ) =q

q + lvsns fp

æ

èç

ö

ø÷

q

• Probability that target DNA is

amplified in at least one aliquot

(detection)

Sensitivity = P Detection | l > 0( ) =1-q

q + lvsns fp

æ

èç

ö

ø÷

qna

Water

sample

Extracted

DNA

PCR

replicate

Amplification

Sampling Scheme

• 95% confidence in detection with varying no. samples, no. PCR replicates and concentration

• At a concentration of 10 molecules/litre, 95% detection probability will be achieved with 10 samples and 5 PCR replicates

na=1

na=2

na=3

na=4

na=10

.

.

.

.

Probability of Detection

Water samples required to achieve 95% confidence in detection

SPRING

0 5 10 15 20 25

0.0

0.4

0.8

Se

nsi

tivity

Carp

0 5 10 15 20 25

0.0

0.4

0.8

Se

nsi

tivity

Weatherloach

0 5 10 15 20 25

0.0

0.4

0.8

Number of samples

Se

nsi

tivity

Redfin

0 5 10 15 20 25

0.0

0.4

0.8

Se

nsi

tivity

Carp

0 5 10 15 20 25

0.0

0.4

0.8

Se

nsi

tivity

Weatherloach

0 5 10 15 20 25

0.0

0.4

0.8

Number of samples

Se

nsi

tivity

Redfin

AUTUMN

European carp control program, Tasmania

100x 600mL samples per lake. 6 qPCR replicates

Lake Sorell Lake Crescent

Lake volume (ML) 120,000 37,000

Carp population estimate 1140-1840 0

Positive eDNA samples 2/100 0/100

Positive qPCR replicates 2/600 0/600

Photo. J. YickPhoto. StudiaPhotos

Lake Sorell Lake Crescent

Lake volume (ML) 120,000 37,000

Carp population estimate 1140-1840 0

Num

ber

of

sam

ple

s

95% detection confidence assuming the current number of carp in the

lake is: Red line, at the lower population estimate (1140 carp) or blue line,

at the upper population estimate (1840 carp)

If 200 carp remain in Lake Sorell,

~7144 x 600mL eDNA samples are

required

Number of carp in Lake Sorell

1. Determined how soon can we detect eDNA after the introduction of the target species.

2. Developed methods for detecting spawning events

3. Investigated parameters for biomass detection of target species

4. Validation of molecular diagnostic assays of marine pests of high priority to Australia

5. Expanded assays for a range of non-fish species, including amphibians, reptiles, molluscs, crustaceans

6. Used high-throughput parallel sequencing to create species inventories from environmental samples

7. Investigating eDNA as tool for biocontrol agent detection

Subsequent studies/applications

http://www.smith-root.com/images/smith-root/edna/edna-process.png

Operationalising eDNA technology

Figure 1

The future of eDNA sampling & processing

Bohan et al. 2017 TREE

Global array of samples,

icloud network

reconstruction

Analysis across

highly replicated

networks

to detect change

Automated sampler

+ sequencing distributed

across global array

SAFA (CSIRO)

Sample Filtration &

Archival

In situ sampling device.

Automated collection of 24

samples, on site for later lab

analysis

MOBI (CSIRO)

Automated Realtime Algal ID

Microbial Oceanography Biosensing Instrument.

Fully automated, in situ instruments for DNA

analysis of water borne microbes.

eDNA for Border Biosecurity

• Commodity importation & real-time detection applications

• e.g Ornamental Fish Trade

Pest & Pathogen diagnostics

Cakechooser.com

Black tetra (Gymnocorymbus ternetzi)

Red belly piranha (Pygocentrus nattereri)

?

Research and Development Needs

• Improve the real-time detection technology

• Develop reference data-bases

• Establish standards and guidelines and QA/QC for application

Email: EcoDNA@Canberra.edu.au @Di_GleesonNZ