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 Belward Campus Drive Rockville, MD USA Phone: Fax: ®Tetracore is a Registered Trademark

EZ-PRRSV™ MPX Belward Campus Drive Rockville, MD Phone: Fax: ®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 Belward Campus Drive Rockville, MD Phone: Fax: ®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 ) NA and EU positive controls and Inhibition Control (IC). Quantification Standards (Cat# TC ) 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 Belward Campus Drive Rockville, MD Phone: Fax: ®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 Belward Campus Drive Rockville, MD Phone: Fax: ®Tetracore is a Registered Trademark IC can be used as Extraction and Inhibition Control. NA and EU Positive Control with IC.

EZ-PRRSV™ MPX Belward Campus Drive Rockville, MD Phone: Fax: ®Tetracore is a Registered Trademark Tetracore In house-Testing (TAMRA Ct values) Data from Collaborator Sample # EZ-PRRSV MPX 4.0 (TC ) 7uL sample/rxn EZ-PRRSV v1 4uL sample/rxn PRRSV NextGen (TC ) 4uL sample/rxn PRRSV NextGen (TC ) 4uL sample/rxn TAMRA Ct values Genetic Group Estimated RNA copy number, according to standard neg. 1j e b c d i *36neg.1e NA / EU * a/NA NA 22.13/ EU 29.10NA 26 / EU 33NA 23 / EU 31NA 23.33NA *neg. 1j h neg. 1f neg.*neg. 1m b NA / EU 20.09NA 34 / EU 24NA 31 / EU b/NA1000 A neg.1f10000 A * g750 unk (8495) *32unk.1munkn 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 Belward Campus Drive Rockville, MD Phone: Fax: ®Tetracore is a Registered Trademark participantsAAAAAAC C AACCEE EE commercial KitKit A Kit B Kit C Kit D MPX 4 RNAEUNAEUNAEUNAEU NA NA+EUEUNA+EUEUNA+EUEU NAEU PRRSV-strainGenotypedilutionCp mean Cp EU Stendal V954EU-110(-2)26.89No Ct26.89No Ct23.27No Ct27.44 No Ct No Ct25.3 EU Stendal V954EU-110(-3)29.97No Ct30.26No Ct26.75No Ct30.67 No Ct No Ct29.2 EU Stendal V954EU-110(-4)33.31No Ct32.96No Ct30.28No Ct33.69 No Ct 38,12 c No Ct 38.72No Ct 36.9 EU Stendal V954EU-110(-5)36.03No Ct36.48No Ct33.42No Ct 35.8 EU Stendal V954EU-110(-6)38,35 c No Ct40,77 c No Ct35,76 c No Ct unknown EU Stendal V1952/97EU-110(-1)24.73No Ct25.22No Ct21.70No Ct25.38 No Ct No Ct22.1 EU Stendal V1952/97EU-110(-2)28.09No Ct28.50No Ct25.43No Ct28.7 No Ct No Ct25.2 EU Stendal V1952/97EU-110(-3)31.46No Ct31.92No Ct28.68No Ct31.96 No Ct 32.03No Ct29.42No Ct29.58 No Ct28.8 EU Stendal V1952/97EU-110(-4)34.21No Ct35.16No Ct31.92No Ct No Ct36.19No Ct32.69No Ct33.82 No Ct31.7 EU Stendal V1952/97EU-110(-5)37.44No Ct39.08No Ct35.40No Ct36.84 No Ct No Ct34.3 ChinaNA-HF10(-2)No Ct24.18No Ct28.03No Ct23.37No Ct No Ct20.52No Ct21.94No Ct 21.8No Ct ChinaNA-HF10(-3)No Ct29.40No Ct31.42No Ct26.76No Ct No Ct23.81No Ct25.73No Ct 24.8No Ct ChinaNA-HF10(-4)No Ct31.69No Ct33.94No Ct29.89No Ct No Ct27.01No Ct28.57No Ct 28.1No Ct ChinaNA-HF10(-5)No Ct36.00No Ct37.83No Ct33.08No Ct No Ct30.84No Ct33.14No Ct 32.2No Ct ChinaNA-HF10(-6)No Ct38.53No Ct 36.36No Ct No Ct33.68No Ct36.71No Ct 34.1No Ct CobbelsdorfEU-11: No Ct27.32No Ct23.92No Ct27.21 No Ct No Ct24.3 Stendal V852EU-11: No Ct26.83No Ct25.83No Ct28.98 No Ct No Ct23.6 Stendal V1904EU-11: No Ct27.82No Ct22.90No Ct26.7 No Ct No Ct24.8 Stendal V1445/99NA1:500No Ct26.09No Ct27.42No Ct25.61No Ct No Ct20.24No Ct22.01No Ct 22.7No Ct USA VD29949/17NA1:500No Ct27.71No Ct28.93No Ct26.47No Ct No Ct21.88No Ct23.60No Ct 21.1No Ct USA 2NA1:500No Ct25.53No Ct26.82No Ct23.77No Ct 22.33No Ct19.21No Ct21.25No Ct 23.6No Ct Aus_LT3EU-21:1029,00 ac No Ct22.12No Ct34.29No Ct No Ct23.1 BY_Bor/59EU (atypical)1:1033,97 ac No Ct29.55No Ct No Ct26.1 BY_231/SZAEU-31:10No Ct 27.46No Ct21.72No Ct24.94 No Ct No Ct21.2 BY_8380/OKT_2pEU-41:10No Ct 29.51No Ct36.14No Ct No Ct27.1 neg. RNA No Ct unknown neg. RNA No Ct unknown RSB 50 No Ct unknown Epizone Ring Trial 2011 Samples retested by Collaborator with EZ-PRRSV™ MPX 4.0

EZ-PRRSV™ MPX Belward Campus Drive Rockville, MD Phone: Fax: ®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 Belward Campus Drive Rockville, MD Phone: Fax: ®Tetracore is a Registered Trademark

EZ-PRRSV™ MPX Belward Campus Drive Rockville, MD Phone: Fax: ®Tetracore is a Registered Trademark 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 will interact with the rope in minutes Pictures and Information courtesy of Dr. J Zimmerman, Iowa State University

EZ-PRRSV™ MPX Belward Campus Drive Rockville, MD Phone: Fax: ®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 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 Belward Campus Drive Rockville, MD Phone: Fax: ®Tetracore is a Registered Trademark Iowa State Oral fluid study Oral fluid samples 90 Vaccinated Pig Serum samples 107 pooled Pig Serum samples

EZ-PRRSV™ MPX Belward Campus Drive Rockville, MD Phone: Fax: ®Tetracore is a Registered Trademark Thank you for your attention