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For veterinary use only. For in vitro use only.
Component Reference Format Color coding
Storage
Upon receipt After initial use
3 - Mix QPCV2 MPEQPCV2 Tube Green -30°C to -10°C -30°C to -10°C
4b – Standard PC. QPCV2 SCQPCV2 Tube Brown -30°C to -10°C -30°C to -10°C
5 - IPC QPCV2 IPCQPCV2 Tube Yellow -30°C to -10°C -30°C to -10°C
o
o
o
Number of cycles Temperature Duration
Step 1 x1 50°C 2 minutes (2’00”)
Step 2 x1 95°C 10 minutes (10’00”)
Step 3 x45 95°C 15 seconds (00’15”)
60°C * 1 minute (01’00”)
Ct Value Interpretation
Fluorescence of the target exceeds the threshold during the
amplification program Ct <45 Detected
Fluorescence of the target does not exceed the threshold during
the amplification program Ct >45 Not detected
o
o
Number of dilutions Number of replicate per dilution Number of independent tests
6 8 3
Number of
pQPCV2 per PCR
Number of positive results per test Number of
positive results /
24 tests
Frequency of
detection (%) Test 1 Test 2 Test 3
32 8 8 8 24 100,0
16 8 8 8 24 100,0
8 7 8 8 23 95,8
4 4 3 3 10 41,7
2 3 5 4 12 50,0
1 1 4 2 7 29,2
Number of
tests (l)
Operators
number
Number of
independent ranges*
(p)
Number of dilutions per
range* (k)
Number of replicate
per dilution (J)
3 1 3 8 1
Level xi (copies
pQPCV2/PCR) x’i
Ct (yi, j)
Test 1 Test 2 Test 3
2.00 E+01 1.30 36.30 36.34 35.92
2.00 E+02 2.30 32.77 32.02 32.29
2.00 E+03 3.30 29.49 28.84 28.63
2.00 E+04 4.30 26.14 25.76 25.35
2.00 E+05 5.30 22.43 22.34 22.05
2.00 E+06 6.30 19.17 19.31 18.91
2.00 E+07 7.30 15.70 15.87 15.51
2.00 E+08 8.30 12.00 12.00 12.47
Slope -3.45 -3.35 -3.34
Intercept 40.83 40.16 39.92
100)110(
1
ae
Parameters Test 1 Test 2 Test 3
Slope (a) -3.45 -3.35 -3.34
Intercept (b) 40.83 40.16 39.92
Linear
regression
coefficient
(r²)
0.9997 0.9986 0.9993
Efficiency (e)
in %94.78 99.02 99.28
Calibration
curve
R² = 0,9998
10,00
15,00
20,00
25,00
30,00
35,00
40,00
0 2 4 6 8 10
Ct
valu
e
Copy number of target per reaction
Quantification range
R² = 0,9986
10,00
15,00
20,00
25,00
30,00
35,00
40,00
0 2 4 6 8 10
Ct
valu
e
Copy number of target per reaction
Quantification range
R² = 0,9993R² = 0,9993
10,00
15,00
20,00
25,00
30,00
35,00
40,00
0 2 4 6 8 10
Ct
valu
e
Copy number of target per reaction
Quantification range
Repeatability of QPCV2 PCR (Ct)
Mean Ct ± Standard deviation CV (%)
P-07-402-10-4 21.9 ± 0.22 1.02
P-07-402-10-5 25.5 ± 0.39 1.55
P-07-402-10-6 29.0 ± 0.28 0.98
P-07-402-10-7 32.7 ± 0.37 1.12
P-07-402-10-8 36.4 ± 0.93 2.55
shows coefficients
of variation (CV) comprised
-0,50
-0,40
-0,30
-0,20
-0,10
0,00
0,10
0,20
0,30
0,40
0,50
0 2 4 6 8 10Me
an b
ias
Concentration of the target x'i (log10/reaction)
Reproducibility of QPCV2 PCR (Ct)
Mean Ct ± Standard deviation CV (%)
P-07-402-10-4 21.9 ± 0.33 1.48
P-07-402-10-5 25.3 ± 0.45 1.78
P-07-402-10-6 29.0 ± 0.36 1.25
P-07-402-10-7 32.6 ± 0.38 1.16
P-07-402-10-8 36.1 ± 0.82 2.27
shows
coefficients of variation (CV) comprised
Critical parameters Robustness QPCV2 PCR (Ct)
Replicate 1 Replicate 2 Replicate 3 Validation
Classical parameter 36.4 37.1 36.6 Yes
Volume of DNA 4.5µL 36.1 36.1 36.7 Yes
5.5µL 36.2 36.8 36.2 Yes
Volume of PCR mix 18µL 36.4 36.5 36.4 Yes
22µL 36.0 35.2 35.7 Yes
Time of hybridization 54 secs 37.6 36.0 36.7 Yes
66 secs 36.1 36.2 35.6 Yes
T°C of hybridization 59°C 36.5 36.7 35.9 Yes
61°C 35.9 37.7 37.8 Yes
QPCV2 PCR stability (Ct)
Lifetime (months) T=0 T=3 T=6 T=9 T=12 T=15
QP
CV
2 L
DP
CR =
8 c
op
/PC
R
Replicate 1 36.3 37.8 36.8
Replicate 2 37.8 37.4 37.6
Replicate 3 38.4 38.0 37.7
Replicate 4 38.3 36.1 36.8
Replicate 5 36.7 36.5 36.9
Replicate 6 36.1 ND 37.1
Replicate 7 37.9 37.1 36.6
Replicate 8 36.5 37.6 38.1
Replicate 9 37.4 37.7 36.8
Replicate 10 36.6 36.3 38.1
GQ
PC
V2
qu
an
tifi
cati
on
ran
ge
SCQPCV2-0P1 9.3 9.8 9.9
SCQPCV2-0P1 10-1 13.2 13.6 13.4
SCQPCV2-0P1 10-2 16.6 17.0 16.9
SCQPCV2-0P1 10-3 20.1 20.3 20.4
SCQPCV2-0P1 10-4 23.6 23.7 23.6
SCQPCV2-0P1 10-5 27.3 27.2 26.9
SCQPCV2-0P1 10-6 30.2 30.3 30.2
SCQPCV2-0P1 10-7 34.5 33.9 33.3
Validation
PENDING
Evaluated method limit of detection
Matrices NucleoSpin® Tissue MagVet™ Universal
Isolation Kit
MagMAX™ Pathogen
RNA/DNA Kit
Serum TS*: 200µL TS: 200µL Ongoing
Oral fluids N/A** TS: 350µL Ongoing
* TS: Test Sample
** N/A: Non Applicable
LDMETHOD copies per mL of serum or oral fluids
Matrices NucleoSpin® Tissue MagVet™ Universal
Isolation Kit
MagMAX™ Pathogen
RNA/DNA Kit
Serum 5.00E+03 cop/ mL 3.00E +03 cop/ mL Ongoing
Oral fluids N/A* 2.00E+03 cop/ mL Ongoing
Status of sample
Positive Negative
Response of the method
Positive TP FP
Negative FN TN
Total TP+FN TN+FP
Note: TP = True Positive / FP = False Positive / TN = True Negative / FN = False Negative
Samples status
(TaqVet™ Porcine Circovirus 2)
Positive
Negative Weak (Ct ≥31)
Medium
(27≤ Ct≤ 31) High (Ct ≤ 27)
Number of tested samples 76 20 13 40
TaqVet™ Porcine Circovirus 2
Positive Negative Total
LSI VetMAX™ Porcine Circovirus
Type 2 – Quantification -
Positive 91 18 109
(73.2 %)
Negative 0 40 40
(26.8 %)
Total 91
(61.1 %)
58
(38.9 %) 149
Samples status
(Internal method of partner lab)
Positive Negative
Weak Medium High
Number of tested samples 54 0 0 126
Samples status
Positive
Negative Quantified,
supportive of
PMWS
Quantified, not
supportive of
PMWS
Not quantified
Number of tested samples 8 50 11 41
1,00E+00
1,00E+01
1,00E+02
1,00E+03
1,00E+04
1,00E+05
1,00E+06
1,00E+07
1,00E+08
1,00E+09
1,00E+10
0 20 40 60 80 100
sam
ple
s co
nce
ntr
atio
n i
n c
op
. P
CV
2/
mL
of
seru
m
Sample ID
Quantification of PCV2 in serum field samples
serum field samples
limit of quantification
PMWS threshold
-1,00
-0,80
-0,60
-0,40
-0,20
0,00
0,20
0,40
0,60
0,80
1,00
4,00 5,00 6,00 7,00 8,00
Ab
so
lute
bia
s
Concentration
absolute bias High limit of tolerance interval
Low limit of tolerance interval high limit of acceptance
Characteristics Validation
Specificity Specific detection of porcine Circovirus type 2 (PCV2)
Efficiency 97.7 % ± 2.53 %
LDPCR 8 copies per PCR
LQPCR 20 copies/ PCR
Repeatability CV < 2.55 %
Intermediate precision CV < 2.27 %
Robustness
Unaffected by variable volume of DNA (+/-10 %), volume of
PCR mix (+/-10 %), time of hybridization (+/-10 %) and T°C of
hybridization (+/-1°)
Stability Validated on 6 months (ongoing on 15 months)
Characteristics Validation
Approach of LDMETHOD
Serum
NucleoSpin® Tissue LDMETHOD = 5.00E+03 cop/ mL
MagVet™ Universal Isolation kit LDMETHOD = 3.00E +03 cop/ mL
MagMAX™ Pathogen RNA/DNA Kit Ongoing
Oral fluids MagVet™ Universal Isolation kit LDMETHOD = 2.00E+03 cop/ mL
MagMAX™ Pathogen RNA/DNA Kit Ongoing
Diagnostic sensitivity - specificity
Serum MagVet™ Universal Isolation kit N/A*
N/A*
Validation of a quantitative method using the accuracy profile
Serum MagVet™ Universal Isolation kit
Domaine de validation = 1.00E+04 à 1.00E+08
cop/ mL
LQMETHOD = 1.00E+04 cop/ mL
Sample reference Origin Nature Detection QPCV kit
Porcine Circovirus Type 2 ISAE Viral culture Detected
PCV2, strain n°2, dil -2 GD Deventer, 2013 Viral culture Detected
PCV2, strain n°2, dil -3 GD Deventer, 2013 Viral culture Detected
PCV2, strain n°2, dil -5 GD Deventer, 2013 Viral culture Detected
PCV2, strain n°1 GD Deventer, 2013 vaccine Detected
Strains Origin Nature Detection QPCV
kit
Virus
Porcine Circovirus Type 1 GD Deventer Viral culture Not detected
Porcine Herpèsvirus 1 (Aujesky) NC* Viral culture Not detected
Porcine Parvovirus NC* Viral culture Not detected
Porcine Influenza H1N1 NC* Viral culture Not detected
Porcine Influenza H3N2 LSI – Life
technologies
Viral culture Not detected
Porcine Influenza H7N1 AHVLA Viral culture Not detected
Porcine Influenza H7N7 AHVLA Viral culture Not detected
Porcine Influenza H1N2 ANSES Ploufragan Viral culture Not detected
PRRSV-EU SYVA Pyrsvac Vaccine Not detected
PRRSV-US Boehringer Ingelheim
Vetmedica GmbH Ingelvac Vaccine
Not detected
African swine fever INIA ref. DNA Kenya Not detected
Classical swine fever ANSES Maison Alfort Viral culture Not detected
Strains Origin Nature Detection
QPCV kit
Bacteria
Mycoplasma hyopneumoniae Not detected
Mycoplasma hyosynoviae Not detected
Mycoplasma hyosynoviae Not detected
Mycoplasma hyosynoviae Not detected
Pasteurella multocida toxinogène Strain 6
Not detected
Brachyspira hyodysenteriae Not detected
Streptococcus suis sérotype VII Not detected
Streptococcus suis sérotype XI Not detected
Streptococcus suis sérotype XIV Not detected
Serum
NucleoSpin® Tissue MagVet™ Universal
Isolation kit
MagMAX™ Pathogen
RNA/DNA Kit
Infection at 5.00E+03 cop
pQPVC2/ mL of serum
Infection at 3.00E+03 cop
pQPVC2/ mL of serum Ongoing
Ct QPCV2 Ct IPC Ct QPCV2 Ct IPC Ct QPCV2 Ct IPC
Session
1
Extraction 1 + (35.8) + (28.2) + (35.8) + (27.1)
Extraction 2 + (37.0) + (28.7) + (34.6) + (27.2)
Extraction 3 + (37.9) + (29.6) + (35.2) + (27.2)
Extraction 4 + (36.9) + (29.1) + (36.0) + (27.4) Ongoing Ongoing
Session
2
Extraction 5 + (36.4) + (29.0) + (37.2) + (27.5)
Extraction 6 + (34.9) + (28.1) + (35.1) + (27.6)
Extraction 7 + (35.2) + (29.0) + (35.1) + (27.6)
Extraction 8 + (35.9) + (29.1) + (35.9) + (27.8)
Oral fluids
MagVet™ Universal Isolation kit MagMAX™ Pathogen RNA/DNA Kit
Infection at 2.00E+03 cop pQPVC2/ mL of
oral fluids Ongoing
Ct QPCV2 Ct IPC Ct QPCV2 Ct IPC
Session
1
Extraction 1 + (34.6) + (27.2)
Extraction 2 + (35.2) + (27.5)
Extraction 3 + (34.7) + (27.3)
Extraction 4 + (34.7) + (27.6) Ongoing Ongoing
Session
2
Extraction 5 + (35.1) + (27.0)
Extraction 6 + (34.7) + (27.1)
Extraction 7 + (36.3) + (27.3)
Extraction 8 + (34.9) + (27.3)
France : 2
Italy: 2
Germany: 9
Austria: 2
Czech Republic: 2
The Netherlands: 4
Thailand: 2
Republic of Korea: 1 LifeTechnologies
Australia : 2
Belarus : 1
Ukraine : 1
#1 PCV-1 strain, 1:5 diluted in negative porcine serum
#2 PCV-2 strain no 2, 102.1 EE/mL, 1:102 diluted in negative porcine serum
#3 PCV-2 strain no 1, 1:103 diluted in negative porcine serum
#4 PCV-2 strain no 1, 1:102 diluted in negative porcine serum
#5 PCV-2 strain no 1, 1:103 diluted in negative porcine serum
#6 PCV-2 strain no 1, 1:105 diluted in negative porcine serum
#7 Negative porcine serum
#8 PCV-2 strain no 1, 1:105 diluted in negative porcine serum
1 PCV-1 strain, 1:5 diluted ND
2 PCV-2 strain no 2, 102.1 EE/mL, 1:102 27
3 PCV-2 strain no 1, 1:103 24.3
4 PCV-2 strain no 1, 1:102 21.4
5 PCV-2 strain no 1, 1:103 23.5
6 PCV-2 strain no 1, 1:105 30.8
7 Negative porcine serum ND
8 PCV-2 strain no 1, 1:105 30.4