Mònica Campàs, Anna Toldrà, Karl B. Andree ... - Vivaldi · Closer to an early arning system Sample Physical state Aquarium OsHV copies/50 ng total DNA Biosensor qPCR 1 dead treated

Mònica Campàs, Anna Toldrà, Karl B. Andree, Edgar Bertomeu, Ana Roque,

Noèlia Carrasco, Ignasi Gairín, Dolors Furones

VIVALDI final meeting – Brest 28/11/19

The objective

Prevent Control Mitigate

To exploit magnetic beads for the

capture and pre-concentration of

ostreid herpesvirus

To develop an electrochemical

biosensor for the detection of ostreid

herpesvirus

Magnetic beads to capture the virus: the strategy

Rapid, simple and cost-effective MB-based viable OsHV-1 capture strategy

Experimental design

April 2017

seawater magnetic

beads

homogenate magnetic

beads

seawater conjugates

homogenate conjugates

24°C, 22h

stomacher

Virus capture and detection

MBs are able to capture the virus from both the

homogenate and the seawater

The viral DNA detected in the conjugates came from virus

particles captured by the MBs

10 0

10 1

10 2

10 3

10 4

10 5

10 6

10 7

To

tal O

sH

V-1

DN

A c

op

ies

W-1 W-2 W-3

To

tal O

sH

V-1

DN

A c

op

ies

0

2000

4000

6000

8000

10000

Virus capture and detection

Homogenate dilutions

MB amount (µL)

0.1 1 10 100 1000

10 2

10 3

10 4

10 5

MBs dilutions

To

tal O

sH

V-1

DN

A c

op

ies

Steric impediments may decrease capture efficiency

MBs were not saturated

Homogenate dilution

1/1000 1/100 1/10 1 10

To

tal O

sH

V-1

DN

A c

op

ies

10 0

10 1

10 2

10 3

10 4

10 5

10 6

10 7

Experimental infections

homogenate

homogenate MBs

seawater

seawater MBs

sterile water

bare MBs

Mortality monitoring, DNA analyses and RNA analyses

~30 oysters/aquarium

intramuscular injection

MB-conjugates

Positive controls

Negative controls

Mortality monitoring

0 2 4 6 8 10

0

20

40

60

80

100

Mo

rtali

ty (

%)

Days post - injection ( dpi )

MB-conjugates are able to infect oysters

MB-conjugates

Positive controls

Negative controls

1 3 5 7 9 11

DNA analyses

Days post-injection (dpi)

1 2 3 4 5 6 7 8 9 10 11

OsH

V-1

DN

A c

op

ies/n

g t

ota

l D

NA

10-1

100

101

102

103

104

105

1

8

4

2

1

11

10


1 2 3 4 5 6 7 8 9 10 11

OsH

V-1

DN

A c

op

ies/n

g t

ota

l D

NA

10-1

100

101

102

103

104

105

4

2

1

1

1

13

2

2


dead/moribund oysters

living oysters


viral DNA loads:

moribund/dead oysters > living oysters

MB-conjugate with the homogenate

RNA analyses

no differences among ORFs

viral gene expression:

moribund oysters > living oysters

Active replication of the virus


MBs are able to capture viable virus particles

moribund living

Ta

rge

t C

t n

orm

aliz

ed

to

EF

1

0

5

10

15

20

25

ORF4

ORF16

ORF42

viral genes

Pre-concentrating agents: homogenate

MBs are able to pre-concentrate OsHV-1 at least 100 times

1 1/10 1/100 1 1/10 1/100

To

tal O

sH

V-1

DN

A c

op

ies

10 0

10 1

10 2

10 3

10 4

10 5

MB + qPCR qPCR

Experimental conditions:

Pre-concentrating agents: seawater

Application to seawater from a depuration experiment (April 2019)

infected oysters

naïve oysters

CONTROL TREATED (HOD SYSTEM)

UP

- no oyster mortality - no OsHV-1 in oysters

Closer to an early arning system

MBs+qPCR qPCR

https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&ved=2ahUKEwjY8On8spvhAhUwx4UKHcnPD9EQjRx6BAgBEAU&url=https://www.maxpixel.net/Tick-Mark-Correct-Yes-Check-Check-Mark-Ok-Green-1292787&psig=AOvVaw1P7_TkHNlmZprpWm2CDGDT&ust=1553538157356102

DNA-based biosensor: the strategy


Amplification technique

Fast Low-cost Simple

Enzyme

Antibody

DNA

Cell

Electrochemical

Optical

Piezoelectric

Thermal

Bio

reco

gnit

ion

e

lem

en

t

Tran

sdu

cer

Sign

al

miniaturised devices

thermal cycling (↑ energy)

in situ analysis

PCR

in situ analysis

constant temperature (↓ energy)

miniaturised devices

Isothermal amplification

Experimental design

TMBred

TMBox

30 min

37 °C

OsHV-1 tailed product

HRP

s gold electrode

C3 stopper

OsHV-1 FwP OsHV-1 RvP

target OsHV-1

e-

OsHV-1 capture probe

reporter probe

RPA product

RPA SHA

10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 0

100

200

300

400

500

Calibration curve and storage stability


LOD biosensor = 207 copies

LOD qPCR = 4 copies

OsHV-1 target copies

Inte

nsit

y (

nA

)

s

Ready-to-use functionalised electrodes

Predicted stability of 3 months at -20 °C

-20°C 4°C

0 7 7 17 17

0

20

40

60

80

100

120

140

Time (days)

Inte

nsit

y (

%)

Overnight

https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&ved=2ahUKEwjY8On8spvhAhUwx4UKHcnPD9EQjRx6BAgBEAU&url=https://www.maxpixel.net/Tick-Mark-Correct-Yes-Check-Check-Mark-Ok-Green-1292787&psig=AOvVaw1P7_TkHNlmZprpWm2CDGDT&ust=1553538157356102


Sample Physical

state Aquarium

OsHV copies/50 ng total DNA

Biosensor qPCR

1 dead treated 3.34 x 10 5 7.13 x 10 5

2 dead treated 4.78 x 10 5 4.81 x 10 5

3 dead treated 7.26 x 10 4 1.79 x 10 5

4 dead treated 6.10 x 10 3 6.52 x 10 3

5 dead treated 5.21 x 10 3 4.38 x 10 3

6 dead treated 2.75 x 10 3 1.98 x 10 3

7 alive treated 1.97 x 10 2 1.21 x 10 2

8 alive treated 5.27 x 10 2 7.83 x 10

9 alive treated 1.50 x 10 2 3.24 x 10

10 alive treated n.d. 1.04 x 10

11 alive treated n.d. 7.93

12 alive control n.d. n.d.





Analysis of oysters


Sample Physical

state Aquarium

OsHV copies/50 ng total DNA

Biosensor qPCR

1 dead treated 3.34 x 10 5 7.13 x 10 5

2 dead treated 4.78 x 10 5 4.81 x 10 5

3 dead treated 7.26 x 10 4 1.79 x 10 5

4 dead treated 6.10 x 10 3 6.52 x 10 3

5 dead treated 5.21 x 10 3 4.38 x 10 3

6 dead treated 2.75 x 10 3 1.98 x 10 3

7 alive treated 1.97 x 10 2 1.21 x 10 2

8 alive treated 5.27 x 10 2 7.83 x 10

9 alive treated 1.50 x 10 2 3.24 x 10

10 alive treated n.d. 1.04 x 10

11 alive treated n.d. 7.93






Analysis of oysters

10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 1

10 2

10 3

10 4

10 5

10 6

10 7

Ele

ctr

och

em

ical b

iosen

so

r

qPCR

Pearson’s r = 0.988; P < 0.001

excellent agreement

Summarising

MBs are able to capture OsHV-1 from both the homogenate and seawater.

Both homogenate and seawater conjugates have the ability to infect oysters.

MBs are able to pre-concentrate virus particles at least 100 times.

MBs are able to pre-concentrate viruses from seawater, being closer to an early warning system.

An electrochemical biosensor for the detection of OsHV-1 has been developed.

The biosensor exhibits good analytical performance, specificity, sensitivity and storage stability.

Publications

CONTACT

This project has received funding from

the European Union’s Horizon 2020

Research and innovation programme

under grant agreement N° 678589

Mònica Campàs

[email protected]

www.vivaldi-project.eu

Follow us on twitter : vivaldieuproj

Follow us on facebook: vivaldiproject
http://www.vivaldi-project.eu/http://www.vivaldi-project.eu/http://www.vivaldi-project.eu/

Documents

Mònica Campàs, Anna Toldrà, Karl B. Andree ... - Vivaldi · Closer to an early arning system Sample Physical state Aquarium OsHV copies/50 ng total DNA Biosensor qPCR 1 dead treated