Transcript Development and Validation of a Novel Real-Time

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