the development and application of novel diagnostic methods for ... · the new assay may be...

The development and application of

novel diagnostic methods for detection

of infectious viral diseases in domestic

animals and in wildlife

Mikael Leijon Head, Division of Bioinformatics and Molecular Biology

Department of Microbiology

National Veterinary Institute

OIE Collaborating Centre

- for Biotechnology-based Diagnosis of

Infectious Diseases in Veterinary

Medicine

The challenge in genetic

detection – the third position

is often undetermined but

variation also exist in the first

(L, S, R) and second (S)

position.

Conventional genetic

detection methods requires

“conserved” regions where

primers and probes can bind.

U C A G

U F F L L

S S S S

Y Y Stop Stop

C C Stop W

U C A G

C L L L L

P P P P

H H Q Q

R R R R

U C A G

A I I I M

T T T T

N N K K

S S R R

U C A G

G V V V V

A A A A

D D E E

G G G G

U C A G

1st

po

siti

on

2nd position

3rd

po

sition

T h e d i l e m m a o f m o l e c u l a r d i a g n o s t i c s

Blue color – 100% sequence conservation

E x e m p e l – N e w c a s t l e D i s e a s e V i r u s ( N D V )

NDV – whole genome

NDV – ”conserved region”

This example illustrates typical sequence variation of an RNA-virus. This is one of the best conserved regions of the NDV genome (nucleotides labeled blue are conserved). Despite this more than a third sequence positions are not conserved (no color). The periodic variation every third position is clearly visible.

E x e m p e l – N e w c a s t l e D i s e a s e V i r u s ( N D V )

NDV – ”conserved region”

S c h e m a t i c v i e w o f t h e m e t h o d

Conventional PCR The new principle

Primer Microorganism genome

Multiplex Primer ”cocktail” that recognize all sequence variants

Pre-amplified microorganism genome

I m p l e m e n t a t i o n f o r A I V - p a t h o t y p n i n g

primer

primer

probe Conventional system

The new system

PCR 1

PCR 2

primer

primer

primer

Cleavage site

Selection primers:

fsHP01 CGGGAACTATCACCAAACAACACCCCGCAAGGAGARAGAARAAGAAAAAAGAG fsLP14 CGGGACAACAAACCACTATCAACCCCGATYCCTCARARAGRAACAARAGG

fsHP02 CGGGAACTATCACCAAACAACACCCCGAAGGGGAGAGAAGAAGAAAAAAGAG fsLP18 CGGGACAACAAACCACTATCAACCCCGTYCCTCARARAGRAACAARAGG

fsHP03 CGGGAACTATCACCAAACAACACCCCGCCCTCAAAGAAAAAGAAAAACAAGAG fsLP19 CGGGACAACAAACCACTATCAACCCCGGTYCCTCARARAGAGACAARAGG

fsHP28 CGGGAACTATCACCAAACAACACCCCGCAAAGAGARAGAAGAARRAAAAGARG fsLP21 CGGGACAACAAACCACTATCAACCCCGGTYCCTCARARAGRTACAARAGG

fsHP29 CGGGAACTATCACCAAACAACACCCCGAAGAGARAGAAGAARRAAGCGARG fsLP26 CGGGACAACAAACCACTATCAACCCCGCCCTCAGAGAGAGACGAGAGG

fsHP06 CGGGAACTATCACCAAACAACACCCCGGTCCCTCAAAGGAAGAAAAGAG fsLP02 CGGGACAACAAACCACTATCAACCCCGCCCTCAACGAGAAACAAGAGG

fsHP30 CGGGAACTATCACCAAACAACACCCCGGAGAKARAAGAAGAAAAAAGMGAGG fsLP03 CGGGACAACAAACCACTATCAACCCCGYCCTCAAARGGAGACAAGAGG

fsHP31 CGGGAACTATCACCAAACAACACCCCGGAGAKARAAGAAGAAAAAAGAGRGG fsLP22 CGGGACAACAAACCACTATCAACCCCGCCTGAAAYTCCAAAAGRAAGG

fsHP32 CGGGAACTATCACCAAACAACACCCCGRGAGAKARAAGAAGAAARAARAGAGG fsLP23 CGGGACAACAAACCACTATCAACCCCGCCTGAAAYTCCAAAAGGAAAAGG

fsHP33 CGGGAACTATCACCAAACAACACCCCGRGAGAKARAAGAAGAAAAARGARAGG fsLP05 CGGGACAACAAACCACTATCAACCCCGYGAAATTCCAAARGGAAGAGG

fsHP37 CGGGAACTATCACCAAACAACACCCCGGGAGAGAGAAGAAGAAAAAAGAGAGG fsLP07 CGGGACAACAAACCACTATCAACCCCGCGAARTYCCAAARGGRAGAG

fsHP38 CGGGAACTATCACCAAACAACACCCCGCCCTCAAAGAAGAAAAAAAAGAGG fsLP08 CGGGACAACAAACCACTATCAACCCCGGAAAYCCCAAAKGGAAGAGG

fsHP08 CGGGAACTATCACCAAACAACACCCCGCCTCAAAGAARAAGAAAAAARAGAGG fsLP10 CGGGACAACAAACCACTATCAACCCCGCYGAARTCCCRAAKGGAAR

fsHP09 CGGGAACTATCACCAAACAACACCCCGGGATCGCGTGTGAGGAG fsLP24 CGGGACAACAAACCACTATCAACCCCGCCTGARAYTCCAAAAGGRAGAG

fsHP10 CGGGAACTATCACCAAACAACACCCCGCCAAAAAAAGGAAAAAAAGAGG fsLP25 CGGGACAACAAACCACTATCAACCCCGCTGARAYTCCAAAGGGRAGAG

fsHP11 CGGGAACTATCACCAAACAACACCCCGGATCGCGSGTGAGGAG fsLP12 CGGGACAACAAACCACTATCAACCCCGCTGAAATTCCAAAAGGAAGAGG

fsHP13 CGGGAACTATCACCAAACAACACCCCGCCAAAGAGGAGGAGGAGAGG fsLP13 CGGGACAACAAACCACTATCAACCCCGAATGTYCCTCAAARAGAAACAAGAG

fsHP14 CGGGAACTATCACCAAACAACACCCCGCCAAAAAAGAGGAAAAAGAGAGG fsLP27 CGGGACAACAAACCACTATCAACCCCGGTTCCTCAAAGAGACACAAGGG

fsHP15 CGGGAACTATCACCAAACAACACCCCGTTCCAAAAAGGAAAAAGAGAGG fsLP29 CGGGACAACAAACCACTATCAACCCCGTCCTCAAAGGGAAACAAGAGG

fsHP34 CGGGAACTATCACCAAACAACACCCCGAAACTCCAAAAAGAAGAARMAGAGG fsLP30 CGGGACAACAAACCACTATCAACCCCGTGGAGTCCCAAGAAAAAGAGG

fsHP17 CGGGAACTATCACCAAACAACACCCCGCCAAAAAAGAAAAAGAAAAAGAGAGG fsLP31 CGGGACAACAAACCACTATCAACCCCGCGGAAAATCCRAAGAMKAGAGG

fsHP35 CGGGAACTATCACCAAACAACACCCCGCCCAAAAAAAGAAGAAARAGAGG fsLP32 CGGGACAACAAACCACTATCAACCCCGCTGAAATCCCAAAGAAAAGAGG

fsHP19 CGGGAACTATCACCAAACAACACCCCGGAAATTCCAAAAAAGAAAAAGAGAGG fsLP33 CGGGACAACAAACCACTATCAACCCCGAGAGAACCCAAAGACCAGAGG

fsHP21 CGGGAACTATCACCAAACAACACCCCGAGAGAGGGAGGAAGAAGAAAAAGAGG fsLP34 CGGGACAACAAACCACTATCAACCCCGTACCAGAAACAGATGACCAGAGG

fsHP22 CGGGAACTATCACCAAACAACACCCCGAAAGAGAGAGAAGGAAAAAGAGAGG fsLP35 CGGGACAACAAACCACTATCAACCCCGCAGAGAAWCCAAAGACCAGAGG

fsHP23 CGGGAACTATCACCAAACAACACCCCGAAAAAAGAAAAAGAAAAAGAGGCC fsLP36 CGGGACAACAAACCACTATCAACCCCGAGAGAABCCMAAGACCAGRGG

fsHP26 CGGGAACTATCACCAAACAACACCCCGCCCGAGAARGAGAAAGAGAGG fsLP37 CGGGACAACAAACCACTATCAACCCCGGARARCCCMAARACCAGAGG

fsHP39 CGGGAACTATCACCAAACAACACCCCGTTCCAAAAAGAGAACAAAAAGAGG fsLP38 CGGGACAACAAACCACTATCAACCCCGCAGAAAATCCAAAAACCAGAGG

fsHP40 CGGGAACTATCACCAAACAACACCCCGAAATCCCAAAGAGAAAGAAAAGAGG fsLP39 CGGGACAACAAACCACTATCAACCCCGARAAWCCAAAGCCCAGAGG

fsHP41 CGGGAACTATCACCAAACAACACCCCGCCCRAAGAAGAGAGAGAAGAGAG fsLP40 CGGGACAACAAACCACTATCAACCCCGAGAGAAACCAAARCCMAGAGG

fsHP42 CGGGAACTATCACCAAACAACACCCCGGCGCAGGCAGAAAAGAGG fsLP41 CGGGACAACAAACCACTATCAACCCCGAGAAAAACCAAAGACAAGGGG

fsHP43 CGGGAACTATCACCAAACAACACCCCGACATGCGCAAAAAAAGAGG fsLP42 CGGGACAACAAACCACTATCAACCCCGCCAGAAAAACCAAAGACAAGAGG

fsHP44 CGGGAACTATCACCAAACAACACCCCGTTCCCCAAAGAGAAAAAAGAGG fsLP43 CGGGACAACAAACCACTATCAACCCCGCAGAGAATCCAAAGACTAGGGG

fsHP45 CGGGAACTATCACCAAACAACACCCCGCCCCAAAGGAAAAAAAGAGG fsLP44 CGGGACAACAAACCACTATCAACCCCGCCCCARARAGRAACAARAGG

fsHP46 CGGGAACTATCACCAAACAACACCCCGGGRGGAAGAAGRAGAAAAAGAGG fsLP45 CGGGACAACAAACCACTATCAACCCCGCCCARARAGRAACAARGGG

fsHP47 CGGGAACTATCACCAAACAACACCCCGCCCCGACCCAGAAGAGG fsLP46 CGGGACAACAAACCACTATCAACCCCGTACCCCAAAGAAAAACAAGAGG

fsHP48 CGGGAACTATCACCAAACAACACCCCGGCCCCAGAAGAAAAAGAGAGG

fsHP49 CGGGAACTATCACCAAACAACACCCCGGTGCMTCAGARGAARAAGAGAGG

fsHP50 CGGGAACTATCACCAAACAACACCCCGGTACCCCAAAGGAAAAAAAGAGG

fsHP51 CGGGAACTATCACCAAACAACACCCCGAGAAAAAGAAAAAGAAAAACAAGAGG

fsHP52 CGGGAACTATCACCAAACAACACCCCGMAKAAACGGATGACCAGAGG

fsHP53 CGGGAACTATCACCAAACAACACCCCGGGAAACGRATGACCAGAGG

fsHP54 CGGGAACTATCACCAAACAACACCCCGGGTGCAGARAAACCAGAGG

fsHP55 CGGGAACTATCACCAAACAACACCCCGAAATTCCAAAAAGAAGAAGGGG

Pre-amplification primers (H5 and H7 specific):

prfH5EH RAAYTTYATTGCTCCAGAAWATGCATAC

prrH5EH TACCAACCGTCTACCATKCCYTG = H5-kha-3: TACCAACCGTCTACCATKCCYTG

prfH5WH TTTATAGCTCCYGAATATGCRTACAA

prrH5WH TACCAYCCRTCHACCATTCCTT

prfH7EH ATGYCCGAGATATGTWAARCA Alt: GK7.4: TTTGTAATCTGCAGCAGTTC

prrH7EH TTTGTAATCTGCHGCAGTYC

prfH7WH.1 GCCCTCGRTATGTCARACA Detection primers:

prfH7WH.2 CCCTCGRTATGTCARGCA fdHP Fam-isoC-CGGGAACTATCACCAAACAACACCCCG

prrH7WH TTTGTARTCAGCTGCAGTYC fdLP CAL orange 560-isoC-CGGGACAACAAACCACTATCAACCCCG

484 PRIMER SYSTEM COVERING ALL AIV

CLEAVAGE SITES TO DATE

I m p l e m e n t a t i o n f o r A I V - p a t h o t y p n i n g

I p r a c t i c e – P a t h o t y p n i n g o f a v i a n i n f l u e n z a

Enhanced cleavability result if the

cleavage site has the motif : R-X-K/R-R

Cleavage- site

Systemic infection with high pathogenicity

Hemagglutinin pro-protein

R-X-K/R-R HA0

HA1 HA2

N e w m e t h o d f o r A I V ( N D V ) p a t h o t y p i n g

Pre-amplification

Multiplex PCR

HP

LP

R e s u l t s f o r c l i n i c a l s p e c i m e n s

Matrix gene amplification (CRL) (Taqman detection – Cy5) H5/H7 amplification (CRL) (No detection)

Pathotyping two-level reaction

The Cepheid SmartCycler

T h e S m a r t C a p ™ S y s t e m

N D V p a t h o t y p i n g o n p o r t a b l e P C R i n s t r u m e n t

C O N C L U S I O N S

This method has the potential to substitute CS sequencing for determination of the molecular pathotype. Rapidly providing crucial information concerning NAI outbreaks which may be critical to their management. The new assay may be extended to the LP/HP differentiation of previously unknown H5/H7 isolates. It may be considered for integration into surveillance and control programmes both in domestic and wild bird populations.

D e t e c t i o n s y s t e m

PCR1

CGGGAACTATCACCAAACAACACCCCGCCATYTGGTTCATGTGGC

Detection primer

19 deg. selection primers

Pre-amplification NS5 gene

FAM

CGGGAACTATCACCAAACAACACCCCG

Selection primers were designed after the

alignment of 1159 whole genome of different

flavivirus viruses WNV/JEV/YFV/Dengue

virus/TBE/St.Louis EV/ USUTU V/Powassan

virus

PCR 2

Primers for PCR1 were from Moureau G. et al 2007 with modification

http://intranet.sva.se/

F l a v i v i r u s d e t e c t i o n

-0,10

-0,08

-0,06

-0,04

-0,02

0,00

0,02

0,04

0,06

1 6 11 16 21 26

Flu

ore

sce

nce

(Fa

m)

Cycle number

Chickungunya Mallaysia

Chickungunya strain 23161

WNV Egypt

WNV Israel

WNV Uganda

WNV MgAn 786

YF Asibi

TBE Hochosterwitz

TBE Sofjin

TBE Latvia

JEV Nakayama

JEV Vietnam

Den 1 Hawaii

Den 1 WP

Den 2 NGC

Den 3 H87

Den 4 H241

NTC

S e n s i t i v i t y o f t h e a s s a y u s i n g W N V P a n e l f r o m Q C M D

-0,08

-0,06

-0,04

-0,02

0,00

0,02

0,04

0,06

0,08

1 6 11 16 21 26 31

Flu

ore

sce

nce

(Fa

m)

Cycle number

WNV NY99 1.2x10E7

WNV NY99 1.2x10E6

WNV NY99 1.2x10E5

WNV NY99 1.2x10E5*

WNV NY99 1.2x10E4

WNV NY99 1.2x10E3

WNV Heja 7.3x10E6

WNV Heja 7.3x10E5

WNV Ug37 1.1x10E5

Non-WNV Flaviviruses 1x10E6

Non-WNV Flaviviruses 1x10E6**

WNV negative

NTC

C o n c l u s i o n

• This novel assay is flexible, simple and valuable for the detection of high

variable RNA viruses

• The assay has been used for pathotyping, charactertization and broad

detection

• The assay is suitable for rapid screening applications.

Evaluation of a portable PCR platform for ASF during outbreaks in an endemically infected population

Liu L, LeBlanc N, Atim S, Chenais E, Esau M, Mwebe R, Nantima N, Ademun AR, Ayebazibwe C, Ståhl K National Veterinary Institute, Sweden; National Animal Disease Diagnostics and Epidemiology Center, Uganda

COLLABORATING CENTRE

MAAIF

• 34.9 million people

• 3.03% population growth rate

• Agricultural sector employs about 80%

• 4.26 million pigs, rapidly growing pig production

• ASF considered a main constraint and porcine cysticercosis and other parasitic diseases

Source: UBOS census, 2014

Uganda

MAAIF

http://en.wikipedia.org/wiki/Kampala

• More pigs in Central, western and eastern Uganda

• Fewer pigs in pastoral areas & Northern Uganda

• Most Pigs in Uganda are kept by smallholder

farmers under backyard system.

MAAIF

Population density of pigs in Uganda

• ASF endemic in Uganda

• Very few outbreaks are reported

• June 2013 to Jan 2016, 41 outbreaks

reported in 14 districts

• Even fewer are lab confirmed

• Resources for surveillance limited

• Inadequate infrastructure for sample

storage and shipping

• Gap between field and central lab

*Muhangi et al 2015, Chenais et al 2015 a,b

MAAIF

Field study carried out April-May 2016

to evaluate the feasibility and performance of a diagnostic system including sample preparation and real-

time PCR assays on a portable thermocycler, for the detection of ASFV on-site during outbreak

investigations or in a simple lab setting in remote areas.

Active out break sites identified through workshop with DVOs

MAAIF

• Five outbreak sites identified and visited

• Three with active outbreaks and clinically affected pigs

• Initial diagnosis on-site and confirmatory diagnosis at NADDEC

MAAIF

Equipment

• Magnet

• A set of pipettes

Sample prep

• Magnetic bead extraction

- Magmax

- Diasorin

• Dilution with PBS

PCR on-site or simple lab

• TCOR8

• Tetracore ASF with IC

• ASF UPL

PCR NADDEC

• Tetracore ASF on TCOR8

• ASF UPL on Mx3000 MAAIF

On-site diagnosis

Platform simple to use in the field. Sample prep remains the bottle

neck.

PCR+ extraction (6 samples)≈2h10 PCR+dilution (6 samples)≈1h30

Dilution 40x

MAAIF

PigID Preparation Volume/ fold Sample type Ct ASF Ct for IC Kit code

no. 1-6 pooled 1/10 blood 0 0 Tetracore

no. 1-6 pooled 1/20 blood 0 0 Tetracore

no. 1-6 pooled 1/40 blood 25.6 0 Tetracore

no. 1-6 pooled 1/80 blood 27.3 0 Tetracore

no. 1-6 pooled 1/10 blood 0 NA ASF UPL

no. 1-6 pooled 1/20 blood 33.6 NA ASF UPL



no. 1-6 magmax 50ul blood 22.8 0 Tetracore

On-site diagnosis - dilution

• Dilution a promising sample prep method, should be with IC

• Inhibition a potential problem

• The IC very valuable MAAIF

Tetracore vs UPL

PigID Diluted in blood

Preparation Volume/fold UPL on

Mx3000p

Tetracore on TCOR8

Ct for IC on TCOR8

2 10 Extraction 20µl 37.76 31.5 30.5 2 10 Dilution 40x 39.81 No Ct No Ct 2 100 Extraction 20µl No Ct 36.1 32.4 2 100 Dilution 40x No Ct No Ct No Ct 2 1000 Extraction 20µl 42.16 36.5 35.2 2 1000 Dilution 40x No Ct No Ct No Ct 3 10 Extraction 20µl 30.06 26 No Ct 3 10 Dilution 40x 34.18 32.8 No Ct 3 100 Extraction 20µl 34.63 No Ct No Ct 3 100 Dilution 40x 38.57 No Ct No Ct 3 1000 Extraction 20µl 36.99 33.4 32.4 3 1000 Dilution 40x 41.46 31 No Ct 7 10 Extraction 20µl 29.93 30.5 No Ct 7 10 Dilution 40x 33.04 28.2 No Ct 7 100 Extraction 20µl 33.64 29 30.2 7 100 Dilution 40x 37.38 29.7 No Ct 7 1000 Extraction 20µl 35.70 31.7 30.2 7 1000 Dilution 40x 40.09 No Ct No Ct

13 10 Extraction 20µl 28.99 25.2 No Ct 13 10 Dilution 40x 31.88 24.9 No Ct 13 100 Extraction 20µl 31.37 29.8 No Ct 13 100 Dilution 40x 35.85 29.2 No Ct 13 1000 Extraction 20µl 34.33 28.5 29.4 13 1000 Dilution 40x 38.66 No Ct No Ct 18 10 Extraction 20µl 38.46 31.9 30.7 18 10 Dilution 40x 41.20 No Ct No Ct 18 100 Extraction 20µl 44.36 36.1 32.5 18 100 Dilution 40x No Ct 32.6 29.7 18 1000 Extraction 20µl 41.38 39.5 36.6 18 1000 Dilution 40x No Ct No Ct No Ct

• Good agreement between the

assays

• Disagreement mostly seen in

diluted samples

• Possibly related to inhibition

• UPL may be slightly more

sensitive, possibly less prone to

inhibition (smaller sample

volumes)

MAAIF

1/10 1/100 1/1000

38

40

0

0

42

0

32

0

36

0

37

0

31

0

32

0

35

0

EX

TR

AC

TIO

N

DIL

UT

ION

EX

TR

AC

TIO

N

DIL

UT

ION

EX

TR

AC

TIO

N

DIL

UT

ION

PIGID: 2

MX3000P TCOR8 TCOR8 - IC

1/10 1/100 1/1000

30

34

35

39

37

41

26

33

0

0

33

31

0

0

0

0

32

0

EX

TR

AC

TIO

N

DIL

UT

ION

EX

TR

AC

TIO

N

DIL

UT

ION

EX

TR

AC

TIO

N

DIL

UT

ION

PIGID: 3

MX3000P TCOR8 TCOR8-IC

30

33

34

37

36

40

31

28

29

30

32

0

0

0

30

0

30

0

EX

TR

AC

TIO

N

DIL

UT

ION

EX

TR

AC

TIO

N

DIL

UT

ION

EX

TR

AC

TIO

N

DIL

UT

ION

PIGID: 7


1/10 1/100 1/1000

1/10 1/100 1/1000

29

32

31

36

34

39

25

25

30

29

29

0

0

0

0

0

29

0

EX

TR

AC

TIO

N

DIL

UT

ION

EX

TR

AC

TIO

N

DIL

UT

ION

EX

TR

AC

TIO

N

DIL

UT

ION

PIGID: 13


PigID District Sample

type Ct ASF Ct IC Extraction kit

44 Busia blood 24.2 0 Magmax extraction


46 Busia blood 0 33.4 Magmax extraction





51 Busia blood 0 34 Magmax extraction

52 Busia blood 23 0 Magmax extraction



44 Busia blood 19.9 0 Diasorin extraction


46 Busia blood 0 32.3 Diasorin extraction



49 Busia blood 0 34 Diasorin extraction






Simple lab setting - comparison extraction kits

• 100% concordant results

• Diasorin doesn’t require cool chain

• but requires heating step…

MAAIF

Wild pig testing

MAAIF

Conclusions

The portable platform is simple to use and could provide a valuable tool

for sensitive and rapid diagnosis in remote areas or areas with poor

surveillance infrastructure such as in Uganda.

However,

• Sample prep remains the bottle neck.

- For on-site use, time and simplicity is critical. Direct PCR on

diluted samples promising, but inhibition & inconsistency still a

concern. In next step, we´ll try simple filter with dilution buffer

sample preparation.

• For use on-site and in simple lab, the stability of reagents is crucial.

Cold chain often unreliable. No cold chain required for PCR kit from

Tetracore and extraction kit Diasorin

• Although on-site use is feasible, turn-around time will be a concern

if several outbreaks are investigated. Often, a simple regional lab

may be more appropriate than testing at each site. Running PCR

instrument in vehicle while travelling between sites was tested

successfully.

MAAIF

Acknowledgements

COLLABORATING CENTRE

• OIE twinning Project between SVA ,

Sweden and NADDEC, Uganda

• EU project Link-TAD

• Prof. Charles Masembe, Wellcome Trust

MAAIF

the development and application of novel diagnostic methods for ... · the new assay may be...

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