User-friendly Microarray for Porcine Respiratory Disease...

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Porcine Respiratory Disease ComplexPorcine Respiratory Disease Complex

Aruna Ambagala

Canadian Food Inspection Agency -Lethbridge Laboratory

National Centres For Animal DiseaseLethbridge, Alberta

• Pneumonia in finishing and fattening pigs

• The most important health concern for swine producers today

• Clinical signs – slow growth, lethargy, anorexia, fever, cough & dyspnea

• A multifactorial disease

• Caused by the interactions of several pathogens, environmental,

management and genetic factors

Porcine Respiratory Disease Complex (PRDC)Porcine Respiratory Disease Complex (PRDC)

Stress, Damage to respiratory tract

Polymicrobial Diseases. Brogden KA, Guthmiller JM, editors. 2002.


• Severity dependents on - The number/type of pathogens involved- Management/environment factors

- Genetic background

Primary AgentsSecondary Agents

• Early detection is critical for controlling the disease to minimize losses

• Current diagnostic tests - Targets one agent at a time- Time consuming & expensive

� The main objective: To develop a rapid molecular assay for PRDC that can identify multiple pathogens.


1. Identify the target genes and create a sequence database

2. Design primers and probes

3. Multiplex PCR - To amplify multiples pathogens

4. DNA Microarray - To detect, differentiate & subtype pathogens

Viruses:

• Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)- # 1 pathogen detected

• Swine Influenza Virus – second most common viral agent• Porcine Circovirus Type 2 (PCV2) – increase severity?• Porcine Respiratory Coronavirus (PRCV) – replicates in epithelium

of lower RT

Bacteria:

• Pasteurella multocida* - The most common bacterial species• Mycoplasma hyopneumoniae – The second most common• Salmonella enterica Choleraesuis*• Streptococcus suis*

* Opportunistic pathogens


Virus Gene # Probes

• Swine Influenza virus Matrix 4• Porcine Circovirus 2 Capsid 8• PRRSV Nucleocapsid 9

• PRCV Spike gene 4

Bacteria Gene # Probes

• Streptococcus suis Suilysin (Cell membrane) 6• P. multocida KMT1 4

Tox-A 2• S. enterica Choleraesuis SC4352 (Metabolic island) 5

• M. hyopneumonia Intergenic space (IG) 4

� Target genes identified, sequence database created, primers and probes identified

Identify the target genes and create a sequence database

Multiplex RTMultiplex RT--PCR Reaction & Amplification Conditions PCR Reaction & Amplification Conditions

Reaction components (25 µl):1X PCR buffer

1 mM of each primer

1 unit of SSIII Pl-Taq Polymerase

1 µl Template (1/50 dilution)

Cycling Conditions:

Temp oC) Time

RT 60 15 min

94 2 min

AMP 94 30 sec

50 15 sec

72 45 sec

72 7 min

4 Hold

• Total PCR Time: 1 hr 20 min

• Works for Bacteria and Viruses – Split-multiplex

X30

PCV-2 PRRSV

SplitSplit--Multiplex Multiplex vsvs SingleplexSingleplex PCRPCR

Extraction - Qiagen, Template: 1µl, [Primer] = 1mM, Rxn vol – 25µl

Amplicons resolved on QIAxcel

100 10-5 NT

C

100 10-5 NT

C

NT

C(n=10) (n=2) (n=2) (n=10)Multiplex MultiplexSingleplex Singleplex

100 10-5 NT

C

100 10-5 NT

C

PRRSV P 2.5

PRRSV P-Vacc

PCV2

PCV 1

H1N1

PRCV 1:10

PRCV 1 1:25

PRCV 1 1:50

S. suis 1

S. entrica choleraesuis

P. multocida

M. hyopneumoniaea

Viru

s

(10 p

rimers

)

Am

plific

atio

n o

f Viru

s a

nd

Bacte

rial T

arg

ets

in a

Sp

lit-Mu

ltiple

x P

CR

Am

plific

atio

n o

f Viru

s a

nd

Bacte

rial T

arg

ets

in a

Sp

lit-Mu

ltiple

x P

CR

KM

T1

Su

ilysy

n

SC

4352

IG

Viru

sB

ac

teria

PRRSV P 2.5

PRRSV P-Vacc

PCV2

PCV 1

H1N1

PRCV 1:10

PRCV 1 1:25

PRCV 1 1:50

S. suis 1


P. multocida

M. hyopneumoniaea

NTC

PRRSV P 2.5

PRRSV P-Vacc

PCV2

PCV 1

H1N1

PRCV 1:10

S. suis 1


P. multocida

M. hyopneumoniaea

NTC

(10 p

rimers

)(1

2 p

rimers

)

Aeromonas hydrophila Z22

NTC

Enterococcus faecalis ATCC 29212

Bacillus cereus ATCC 14579

Pseudomonas aeruginosa ATCC 27853

Klebsiella pneumoniae ATCC 13883

E. coli ATCC 25922

M. hyorhinis

M. pneumoniae

M. bovis

To

x-A

KM

T1

Sp

litS

plit- -M

ultip

lex P

CR

M

ultip

lex P

CR

– –K

MT

1 a

nd

K

MT

1 a

nd

To

xT

ox

- -A S

pecific

ity

A S

pecific

ity

(12 p

rimers

)

1059

4533 (Tox)

4837 (Tox)

Ser A

Ser B

Ser D

(Ext) - 10-1291

Pm1

Pm2

Pm3

Pm4

Pm5

M. haemolytica Z13

P. m

ulto

cid

a

Print Probes on Microarray

Design Probes & Primers

(Bioedit & AlleleID)

Identify Signatures

Hybridize, Wash

& Report

Fluorescence

intensity

Capture probe

(18-40 bp, Tm: 40-70oC)

Reporter probe

Clinical Sample

DNA/RNA

extraction

Amplification

PCR/RT-PCR

DNA Microarrays DNA Microarrays –– for Detection & Typing of Pathogens for Detection & Typing of Pathogens

Slide Microarray - Passive hybridization - ON

- Labour intensive

Build a Sequence Database

PCR Amplicon

P/N ratio

NC400 Electronic MicroarrayNC400 Electronic Microarray

• Electrophoretically driven printing and hybridization (sec vs hrs)

• An amplicon-to-answer system

• All functions are programmed to run automatically

• Open platform: Can print probes on site

• 400 independently activated test sites: High throughput

• Fast (30 min), User-friendly

Heat Maps: Visualize the microarray data. Bright red - the strong signal (P/N), dull red for weak signal

PRRSV NA (2)

PRRSV EU (2)

Virus

PRRSV-NA( 7 strains) P

RR

SV

EU

SIV

PC

V2

PR

CV

TG

EV

*D

en

gu

e

NT

C

SIV (2)

PCV (3)

PRCV (1)Dengue (1)

NSP

PRRSV (1)

Pro

be

s

• PRCV is a spike (S) gene deletion mutant of TGEV*

• The PRCV probe does not recognize TGEV

• NSP = Non Specific Probe


Heat Maps: Visualize the microarray data. Bright red - the strong signal (P/N), dull red for weak signal

PRRSV NA (2)

PRRSV EU (2)

Virus

PRRSV-NA( 7 strains) P

RR

SV

EU

SIV

PC

V2

PR

CV

TG

EV

*D

en

gu

e

NT

C

SIV (2)

PCV (3)

PRCV (1)Dengue (1)

NSP

PRRSV (1)

Pro

be

s


• + - Negative controls for specificity

• Toxigenic strains of P. multocida - type 0 (4533)- 4837

Bacteria

S.

su

is

S.

py

og

en

es

+

P.

mu

lto

cid

a

P.

ha

em

oly

tica

+

S.e

. C

ho

lera

es

uis

S.e

. e

nte

riti

dis

+M

. h

yo

pn

eu

mo

nia

e

M.

pn

eu

mo

nia

e+

De

ng

ue

NT

C

S. suis Suilysyn (2)

P. Multocida KMT1 (2)

P. multocida Tox-A (2)

S. enterica Choloraesuis (2)

M. hyopneumoniae (2)

Dengue (1)NSP

• PRCV is a spike (S) gene deletion mutant of TGEV*

• The PRCV probe does not recognize TGEV

• NSP = Non Specific Probe

• A fully-automated sample-to-answer system (extraction to detection)

• Low cost disposable cards

• Microfluidic System: Pumps, valves, microchannels, reservoirs & reaction chambers

• 4 samples per card; 36 X4 (144) probe sites per card

• Machine can run 6 cards at a time (24 samples at a time)

Encompass Encompass MDxMDx SystemSystem

3 ½”3

½”

Clinical sample added

DNA/RNA extraction

Divide into two chambers

Two independent PCR

Added into the same compt

Reverse dot blot using HRP

1 2 3 4 5 6 7 8 9

A

B

C

D NDV-HPAIV-1PRRS -7 EU

PRRSV-3

SIV-1M. h2Sec1Pm4Sts3

NDV-2Deng -2PRRS -6 COM

PRRSV-2

PCV2-3M. h1PmT3Pm3Sts2

NDV-1Deng -1PRRS -5 COM

PRRSV-1

PCV2-2Sec3PmT2Pm2Sts1

AIV-2PCoV-1PRRS -4 EU

SIV-2PCV2-1Sec2PmT1Pm1Spot. Control

1 2 3 4 5 6 7 8 9

A

B

C

D

P. multocida

1 2 3 4 5 6 7 8 9

A

B

C

D

PRRSV-NA

1 2 3 4 5 6 7 8 9

A

B

C

D

PCV2

Encompass Encompass MDxMDx System: Preliminary DataSystem: Preliminary Data

Spotting Matrix

• Samples: Porcine Nasal swab material

spiked with virus or bacteria and internal control

Dengue

• C1 – spotting control or dengue virus probe?

SummarySummary

• Created a sequence database of targeted genes for the most important viral and bacterial pathogens of PRDC

• Two multiplex PCR were developed – Virus (10 primers) , Bact (12 primers)

• Sensitivity comparable to singleplex PCR assays

• Tested two different automated detection platforms

- Amplicon-to-answer system (NC400)

- Sample-to-answer system (Encompass MDx system)

• Both systems show excellent results

Future WorkFuture Work

• Continue to test more laboratory samples

• Test the clinical samples from MAFRI to compare the sensitivity and specificity

• Transfer the technology to MAFRI and other laboratories interested

Acknowledgments

Virology group - CFIA Lethbridge Laboratory

• Dr. Oliver Lung

• Dr. Sam Ohene-Adjei*

• Tara Furukawa-Stoffer

Dr. Tomy Joseph - Vet. Diagnostic Services, Agriculture Food & Rural Initiatives,

Winnipeg, MB

Dr. Robin King - Food Safety and Animal Health Division, Alberta Agriculture

& Rural development, Edmonton, AB.

Thank you!

User-friendly Microarray for Porcine Respiratory Disease...

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