EZ-PRRSV™ MPX 4.0 Development and Validation of a Novel Real-Time RT-PCR Assay to Distinguish Porcine Reproductive and Respiratory Syndrome (PRRS) Virus Subtypes with High Sensitivity 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 The EZ-PRRSV™ MPX 4.0 test features: 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. 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. 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 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. 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 Quantification Standards EU PRRSV standard curve (1e5 to 1e1 copies/µl) TAMRA Channel, ABI 7500 NA PRRSV standard curve (1e5 to 1e1 copies/µl) FAM Channel, ABI 7500 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 IC can be used as Extraction and Inhibition Control. NA and EU Positive Control with IC. 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 Tetracore In house-Testing Comparison study of EZPRRSV™ 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 Data from Collaborator (TAMRA Ct values) PRRSV NextGen (TC-9039-096) 4uL sample/rxn TAMRA Ct values Sample # EZ-PRRSV MPX 4.0 (TC9060-096) 7uL sample/rxn EZ-PRRSV v1 4uL sample/rxn PRRSV NextGen (TC9039-096) 4uL sample/rxn Genetic Group Estimated RNA copy number, according to standard 145 27.62 33.5 neg. 146 23.33 28 26 neg. 1j 25000 22.84 1e 147 25.74 31 33 30.95 1b 75000 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 100000 286 21.56 25 23 26.04 1b 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. 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 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 EU RNA EU NA EU NA EU NA EU NA NA+EU EU NA+EU EU NA+EU EU NA 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 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 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 PRRSV-strain Aus_LT3 BY_Bor/59 Epizone Ring Trial 2011 Samples retested by Collaborator with 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 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. 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 EZ-PRRSV™ MPX 4.0 Oral fluid collection from pigs 1. Use cotton rope 2. Adjust length to pig size 3. Extract fluid from rope 4. Pour fluid into a tube chill or freeze 5. Submit to lab for testing ±75% of pigs in a pen of 25-30 will interact with the rope in 20-30 minutes Pictures and Information courtesy of Dr. J Zimmerman, Iowa State University 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 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 Ran # Recommended Extraction Method (magnetic beads) Extraction Method 1 (magnetic beads) 200 µl sample Extraction Method 2 Extraction Method 3 (column) (magnetic beads) 140 µl sample 100 µl sample Extraction Method 4 (magnetic beads) 200 µl sample 4001 4002 4003 4004 4005 4006 4007 4008 4009 4010 4011 0.0 33.3 33.8 35.8 0.0 34.6 32.0 33.6 30.8 37.4 31.1 0 0.0 0.0 0.0 0.0 35.1 32.8 33.2 34.8 35.4 37.0 33.1 33.9 37.1 35.9 36.8 35.6 36.1 0.0 36.8 0.0 0.0 0.0 0.0 0.0 38.8 32.9 34.1 37.0 36.6 34.2 31.0 31.8 34.5 33.2 35.2 33.9 34.3 36.2 35.6 34.0 31.2 31.7 34.0 33.5 0.0 34.5 35.2 34.8 33.9 33.0 31.5 32.3 35.9 33.9 9901 Belward Campus Drive • Rockville, MD 20850 • Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark Extraction Method 5 (column) 200 µl sample 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 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 Thank you for your attention 9901 Belward Campus Drive • Rockville, MD 20850 • Phone: 240-268-5400 • Fax: 240-268-1107 ®Tetracore is a Registered Trademark