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