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
9901 Belward Campus Drive • Rockville, MD 20850 • Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark
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
9901 Belward Campus Drive • Rockville, MD 20850 • Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark
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
9901 Belward Campus Drive • Rockville, MD 20850 • Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark
IC can be used as Extraction and Inhibition Control.
NA and EU Positive Control with IC.
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
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
9901 Belward Campus Drive • Rockville, MD 20850 • Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark
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(-3) No Ct 29.40 No Ct 31.42 No Ct 26.76 No Ct 26.82 27.15 No Ct 23.81 No Ct 25.73 No Ct 24.8 No Ct
China NA-HF 10(-4) No Ct 31.69 No Ct 33.94 No Ct 29.89 No Ct 29.68 29.54 No Ct 27.01 No Ct 28.57 No Ct 28.1 No Ct
China NA-HF 10(-5) No Ct 36.00 No Ct 37.83 No Ct 33.08 No Ct 33.41 33.76 No Ct 30.84 No Ct 33.14 No Ct 32.2 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
9901 Belward Campus Drive • Rockville, MD 20850 • Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark
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
9901 Belward Campus Drive • Rockville, MD 20850 • 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
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
9901 Belward Campus Drive • Rockville, MD 20850 • Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark
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
9901 Belward Campus Drive • Rockville, MD 20850 • Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark
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
9901 Belward Campus Drive • Rockville, MD 20850 • Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark
Thank you for your attention
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