Development and Validation of a Novel Real-Time RT-PCR Assay to Distinguish Porcine Reproductive and...

Development and Validation of a Novel Real-Time RT-PCR Assay to Distinguish Porcine Reproductive and Respiratory

Syndrome (PRRS) Virus Subtypes with High Sensitivity

EZ-PRRSV™ MPX 4.0

9901 Belward Campus Drive • Rockville, MD 20850 USA• Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark

EZ-PRRSV™ MPX 4.0

9901 Belward Campus Drive • Rockville, MD 20850 • Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark

The EZ-PRRSV™ MPX 4.0 test features:

Simultaneous detection and differentiation of NA and EU PRRS viral RNA with Inhibition Control (IC), all in one test.

Improved upon previous Nextgen PRRSV multiplex assay but with new real-time reagents, added primers and probes for broader detection and a larger sample volume.

Broader detection of PRRSV strains by targeting multiple conserved genetic regions of the PRRS virus.

The EZ-PRRSV™ MPX 4.0 test is a real-time RT-PCR diagnostic reagent for the specific detection and differentiation of North American (NA) and European-like/Lelystad (EU) PRRS viral RNA from swine serum, semen, blood, and oral fluid samples.

EZ-PRRSV™ MPX 4.0


What is new?• Total run time of 1:22 h (run time depends on instrument).• Complete differentiation of NA and EU PRRSV genotypes:

• each type has it own reporter dye. • double infections with NA and EU PRRSV can be detected in one

reaction.• Lower price.• Higher tolerance to inhibition factors.• Higher template volume.• Control Set (Cat# TC-9061-096)

• NA and EU positive controls and Inhibition Control (IC).• Quantification Standards (Cat# TC-9062-001)

• Prediluted NA and EU IVT standards with concentrations from 1e5 to 1e2 copies/µl, dilution buffer (if lower concentration standard is desired) and IC.

• The purpose of standards is to create a standard curve for qPCR.

EZ-PRRSV™ MPX 4.0


EU PRRSV standard curve (1e5 to 1e1 copies/µl)TAMRA Channel, ABI 7500

Quantification Standards

NA PRRSV standard curve (1e5 to 1e1 copies/µl)FAM Channel, ABI 7500

EZ-PRRSV™ MPX 4.0


IC can be used as Extraction and Inhibition Control.

NA and EU Positive Control with IC.

EZ-PRRSV™ MPX 4.0


Tetracore In house-Testing (TAMRA Ct values)

Data from Collaborator

Sample #EZ-PRRSV MPX 4.0 (TC-9060-096)

7uL sample/rxn

EZ-PRRSV v1 4uL sample/rxn

PRRSV NextGen (TC-9039-096)

4uL sample/rxn

PRRSV NextGen (TC-9039-096)

4uL sample/rxn TAMRA Ct values

Genetic Group

Estimated RNA copy number, according to

standard

145 27.62 33.5 neg. neg. 1j 25000

146 23.33 28 26 22.84 1e 75000

147 25.74 31 33 30.95 1b 50000

148 22.24 25 26 24.47 1c 2500000

150 24.95 27 26 20.66 1d 10000

152 21.98 26 27 26.29 1i 25000

156 30.22 33.5* 36 neg. 1e 1000

220 NA 31.96 / EU 19.17 23.0* 24 25.7 1a/NA 750

222 NA 22.13/ EU 29.10 NA 26 / EU 33 NA 23 / EU 31 NA 23.33 NA 100000

227 29.47 33.0* neg. neg. 1j 1000

248 20.27 23 22 25.19 1h 750000

251 31.24 34 neg. neg. 1f 7500

278 0.00 neg.* neg. neg. 1m 250

286 21.56 25 23 26.04 1b 100000

362 NA 29.82 / EU 20.09 NA 34 / EU 24 NA 31 / EU 26 28.96 1b/NA 1000

A20 23.07 27 33 neg. 1f 10000

A25 24.02 28* 28 34.01 1g 750

unk (8495) 22.99 27* 32 unk. 1m unkn

Estimated TAMRA Ct value based on Log 1 Ct result.

* 8uL of sample was used.

Comparison study of EZ-PRRSV™ MPX 4.0 and PRRSV NextGen:

• 18 samples from different genetic groups and double infections.

• Samples received from collaborator and extracted in house.

• Different sample volumes/reaction.

• 2 different laboratories

EZ-PRRSV™ MPX 4.0


participants A A A A A A C C A A C C E E E E

commercial Kit Kit A Kit A Kit B Kit B Kit C Kit C Kit C Kit C Kit D Kit D Kit D Kit D Kit D Kit D MPX 4 MPX 4

RNA EU NA EU NA EU NA EU NA NA+EU EU NA+EU EU NA+EU EU NA EU

PRRSV-strain Genotype dilution Cp mean Cp mean Cp mean Cp mean Cp mean Cp mean Cp mean Cp mean Cp mean Cp mean Cp mean Cp mean Cp mean Cp mean Cp Cp

EU Stendal V954 EU-1 10(-2) 26.89 No Ct 26.89 No Ct 23.27 No Ct 27.44 No Ct 29.45 29.49 27.48 27.75 28.49 28.72 No Ct 25.3

EU Stendal V954 EU-1 10(-3) 29.97 No Ct 30.26 No Ct 26.75 No Ct 30.67 No Ct 33.25 33.88 31.98 31.98 32.58 35.16 No Ct 29.2

EU Stendal V954 EU-1 10(-4) 33.31 No Ct 32.96 No Ct 30.28 No Ct 33.69 No Ct 38,12c No Ct No Ct No Ct 38.72 No Ct No Ct 36.9

EU Stendal V954 EU-1 10(-5) 36.03 No Ct 36.48 No Ct 33.42 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct 35.8

EU Stendal V954 EU-1 10(-6) 38,35c No Ct 40,77c No Ct 35,76c No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct unknown unknown

EU Stendal V1952/97 EU-1 10(-1) 24.73 No Ct 25.22 No Ct 21.70 No Ct 25.38 No Ct 31.23 27.24 26.76 22.64 30.17 23.54 No Ct 22.1

EU Stendal V1952/97 EU-1 10(-2) 28.09 No Ct 28.50 No Ct 25.43 No Ct 28.7 No Ct 34.43 28.43 31.64 25.62 34.66 26.42 No Ct 25.2

EU Stendal V1952/97 EU-1 10(-3) 31.46 No Ct 31.92 No Ct 28.68 No Ct 31.96 No Ct No Ct 32.03 No Ct 29.42 No Ct 29.58 No Ct 28.8

EU Stendal V1952/97 EU-1 10(-4) 34.21 No Ct 35.16 No Ct 31.92 No Ct 34.76 32.89 No Ct 36.19 No Ct 32.69 No Ct 33.82 No Ct 31.7

EU Stendal V1952/97 EU-1 10(-5) 37.44 No Ct 39.08 No Ct 35.40 No Ct 36.84 No Ct No Ct No Ct No Ct No Ct No Ct 37.04 No Ct 34.3

China NA-HF 10(-2) No Ct 24.18 No Ct 28.03 No Ct 23.37 No Ct 23.22 23.00 No Ct 20.52 No Ct 21.94 No Ct 21.8 No Ct




China NA-HF 10(-6) No Ct 38.53 No Ct No Ct No Ct 36.36 No Ct 36.4 36.76 No Ct 33.68 No Ct 36.71 No Ct 34.1 No Ct

Cobbelsdorf EU-1 1:500 24.28 No Ct 27.32 No Ct 23.92 No Ct 27.21 No Ct 26.95 26.01 23.94 23.69 25.91 24.19 No Ct 24.3

Stendal V852 EU-1 1:500 24.07 No Ct 26.83 No Ct 25.83 No Ct 28.98 No Ct 29.50 28.20 24.67 24.93 28.63 25.86 No Ct 23.6

Stendal V1904 EU-1 1:500 25.26 No Ct 27.82 No Ct 22.90 No Ct 26.7 No Ct 29.81 28.18 27.41 25.59 29.99 27.13 No Ct 24.8

Stendal V1445/99 NA 1:500 No Ct 26.09 No Ct 27.42 No Ct 25.61 No Ct 24.95 23.06 No Ct 20.24 No Ct 22.01 No Ct 22.7 No Ct

USA VD29949/17 NA 1:500 No Ct 27.71 No Ct 28.93 No Ct 26.47 No Ct 25.96 24.93 No Ct 21.88 No Ct 23.60 No Ct 21.1 No Ct

USA 2 NA 1:500 No Ct 25.53 No Ct 26.82 No Ct 23.77 No Ct No Ct 22.33 No Ct 19.21 No Ct 21.25 No Ct 23.6 No Ct

Aus_LT3 EU-2 1:10 29,00ac No Ct 22.12 No Ct 34.29 No Ct No Ct No Ct 24.95 21.77 21.41 20.24 24.24 21.37 No Ct 23.1

BY_Bor/59 EU (atypical) 1:10 33,97ac No Ct 29.55 No Ct No Ct No Ct No Ct No Ct 32.66 28.31 29.67 26.00 33.10 26.60 No Ct 26.1

BY_231/SZA EU-3 1:10 No Ct No Ct 27.46 No Ct 21.72 No Ct 24.94 No Ct 23.77 25.45 21.25 23.08 22.68 24.35 No Ct 21.2

BY_8380/OKT_2p EU-4 1:10 No Ct No Ct 29.51 No Ct 36.14 No Ct No Ct No Ct 28.03 26.25 24.46 24.72 27.52 25.21 No Ct 27.1

neg. RNA No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct unknown unknown

neg. RNA No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct unknown unknown

RSB 50 No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct No Ct unknown unknown

Epizone Ring Trial 2011Samples retested by Collaborator with EZ-PRRSV™ MPX 4.0

EZ-PRRSV™ MPX 4.0


Iowa State University PRRS Oral fluid study 2012(Dr. J Zimmerman and Chris Olsen)

• 90 pigs (40 to 50 pounds) were vaccinated with MLV PRRSV vaccine and placed into a PRRSV negative barn 14 days post vaccination to create pens of 25 pigs each with ‐known PRRSV prevalence of 0, 4%, 12%, 20% and 36%.

• The oral fluid samples were collected then tested by PRRSV RT PCR at 6 cooperating ‐laboratories, and the data was analyzed for the probability of a positive PRRSV RT‐PCR result as a function of:

(1) The number (prevalence) of viremic pigs in a pen.(2) The cumulative time the viremic pigs interact with the rope during a 30 minute

collection period.(3) The level of viremia in the positive pigs.(4) The PRRSV oral fluid PCR protocol and laboratory performing the assay.

EZ-PRRSV™ MPX 4.0


EZ-PRRSV™ MPX 4.0


Oral fluid collection from pigs1. Use cotton rope2. Adjust length to pig size3. Extract fluid from rope4. Pour fluid into a tube chill or freeze5. Submit to lab for testing

±75% of pigs in a penof 25-30 will interactwith the rope in 20-30minutes

Pictures and Information courtesy of Dr. J Zimmerman, Iowa State University

EZ-PRRSV™ MPX 4.0


Ran #Recommended

Extraction Method (magnetic beads)

Extraction Method 1(magnetic beads)

200 µl sample

Extraction Method 2 (column)

140 µl sample

Extraction Method 3 (magnetic beads)

100 µl sample

Extraction Method 4 (magnetic beads)

200 µl sample

Extraction Method 5 (column)

200 µl sample

4001 0.0 0 0.0 0.0 0.0 0.0

4002 33.3 35.1 32.8 33.2 34.8 35.4

4003 33.8 37.0 33.1 33.9 37.1 35.9

4004 35.8 36.8 35.6 36.1 0.0 36.8

4005 0.0 0.0 0.0 0.0 0.0 0.0

4006 34.6 38.8 32.9 34.1 37.0 36.6

4007 32.0 34.2 31.0 31.8 34.5 33.2

4008 33.6 35.2 33.9 34.3 36.2 35.6

4009 30.8 34.0 31.2 31.7 34.0 33.5

4010 37.4 0.0 34.5 35.2 34.8 33.9

4011 31.1 33.0 31.5 32.3 35.9 33.9

Comparison of different extraction methods for Oral fluids with EZ-PRRSV™ MPX 4.0 assay

All samples had to be manually prepared before extraction could be performed

EZ-PRRSV™ MPX 4.0


Iowa State Oral fluid study 2012

• 150 Oral fluid samples

• 90 Vaccinated Pig Serum samples

• 107 pooled Pig Serum samples

EZ-PRRSV™ MPX 4.0


Thank you for your attention

Development and Validation of a Novel Real-Time RT-PCR Assay to Distinguish Porcine Reproductive and...

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