For veterinary use only. For in vitro use only.

Transcription

1 For veterinary use only. For in vitro use only.

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6 Component Reference Format Color coding Upon receipt Storage 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

7 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

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9 Number of dilutions Number of replicate per dilution Number of independent tests Number of pqpcv2 per PCR Number of positive results per test Test 1 Test 2 Test 3 Number of positive results / 24 tests Frequency of detection (%) , , , , , ,2

10 Number of tests (l) Operators number Number of independent ranges* (p) Number of dilutions per range* (k) Number of replicate per dilution (J) Level xi (copies pqpcv2/pcr) x i Ct (yi, j) Test 1 Test 2 Test E E E E E E E E Slope Intercept

11 Ct value Ct value Ct value e (10 1 a 1) 100 Parameters Test 1 Test 2 Test 3 Slope (a) Intercept (b) Linear regression coefficient (r²) Efficiency (e) in % ,00 Quantification range 40,00 Quantification range 40,00 Quantification range 35,00 35,00 35,00 30,00 30,00 30,00 Calibration 25,00 25,00 25,00 curve 20,00 20,00 20,00 15,00 15,00 15,00 R² = 0,9998 R² = 0,9986 R² = 0, , , , Copy number of target per reaction Copy number of target per reaction Copy number of target per reaction

12 Mean bias 0,50 0,40 0,30 0,20 0,10 0,00-0, ,20-0,30-0,40-0,50 Concentration of the target x'i (log10/reaction) Repeatability of QPCV2 PCR (Ct) Mean Ct ± Standard deviation CV (%) P ± P ± P ± P ± P ± of variation (CV) comprised shows coefficients

13 Reproducibility of QPCV2 PCR (Ct) Mean Ct ± Standard deviation CV (%) P ± P ± P ± P ± P ± coefficients of variation (CV) comprised shows

14 Critical parameters Robustness QPCV2 PCR (Ct) Replicate 1 Replicate 2 Replicate 3 Validation Classical parameter Yes Volume of DNA 4.5µL Yes 5.5µL Yes Volume of PCR mix 18µL Yes 22µL Yes Time of hybridization 54 secs Yes 66 secs Yes T C of hybridization 59 C Yes 61 C Yes

15 GQPCV2 quantification range QPCV2 LDPCR = 8 cop/pcr QPCV2 PCR stability (Ct) Lifetime (months) T=0 T=3 T=6 T=9 T=12 T=15 Replicate Replicate Replicate Replicate Replicate Replicate ND 37.1 Replicate Replicate Replicate Replicate SCQPCV2-0P SCQPCV2-0P SCQPCV2-0P SCQPCV2-0P SCQPCV2-0P SCQPCV2-0P SCQPCV2-0P SCQPCV2-0P Validation PENDING

16 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

17 Matrices NucleoSpin Tissue LDMETHOD copies per ml of serum or oral fluids MagVet Universal Isolation Kit MagMAX Pathogen RNA/DNA Kit Serum 5.00 E +03 cop/ ml 3.00 E +03 cop/ ml Ongoing Oral fluids N/A* 2.00 E +03 cop/ ml Ongoing Status of sample Positive Negative Positive TP FP Response of the method Negative FN TN Total TP+FN TN+FP Note: TP = True Positive / FP = False Positive / TN = True Negative / FN = False Negative

18 Samples status (TaqVet Porcine Circovirus 2) Weak (Ct 31) Positive Medium (27 Ct 31) High (Ct 27) Negative Number of tested samples LSI VetMAX Porcine Circovirus Type 2 Quantification - TaqVet Porcine Circovirus 2 Positive Negative Total Positive Negative 0 40 Total 91 (61.1 %) 58 (38.9 %) 109 (73.2 %) 40 (26.8 %) 149 Samples status (Internal method of partner lab) Positive Weak Medium High Negative Number of tested samples

19 samples concentration in cop. PCV2/ ml of serum Positive Samples status Quantified, supportive of Quantified, not supportive of Not quantified Negative PMWS PMWS Number of tested samples Quantification of PCV2 in serum field samples 1,00E+10 1,00E+09 1,00E+08 1,00E+07 1,00E+06 1,00E+05 1,00E+04 1,00E+03 serum field samples limit of quantification PMWS threshold 1,00E+02 1,00E+01 1,00E Sample ID

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21 Absolute bias absolute bias High limit of tolerance interval Low limit of tolerance interval high limit of acceptance 1,00 0,80 0,60 0,40 0,20 0,00-0,204,00 5,00 6,00 7,00 8,00-0,40-0,60-0,80-1,00 Concentration

22 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)

23 Characteristics Validation Approach of LDMETHOD Serum Oral fluids NucleoSpin Tissue LDMETHOD = 5.00 E +03 cop/ ml MagVet Universal Isolation kit LDMETHOD = 3.00 E +03 cop/ ml MagMAX Pathogen RNA/DNA Kit Ongoing MagVet Universal Isolation kit LDMETHOD = 2.00 E +03 cop/ ml MagMAX Pathogen RNA/DNA Kit Ongoing Diagnostic sensitivity - specificity Serum MagVet Universal Isolation kit Validation of a quantitative method using the accuracy profile N/A* N/A* Serum MagVet Universal Isolation kit Domaine de validation = 1.00 E +04 à 1.00 E +08 cop/ ml LQMETHOD = 1.00 E +04 cop/ ml

24 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 Virus Strains Origin Nature Detection QPCV 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 LSI Life Viral culture Not detected Porcine Influenza H3N2 technologies 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 kit Strains Origin Nature Mycoplasma hyopneumoniae Mycoplasma hyosynoviae Mycoplasma hyosynoviae Mycoplasma hyosynoviae Detection QPCV kit Not detected Not detected Not detected Not detected Bacteria Pasteurella multocida toxinogène Brachyspira hyodysenteriae Strain 6 Not detected Not detected Streptococcus suis sérotype VII Not detected Streptococcus suis sérotype XI Not detected Streptococcus suis sérotype XIV Not detected

25 Serum NucleoSpin Tissue Infection at 5.00 E +03 cop pqpvc2/ ml of serum MagVet Universal Isolation kit Infection at 3.00 E +03 cop pqpvc2/ ml of serum MagMAX Pathogen RNA/DNA Kit Ongoing Ct QPCV2 Ct IPC Ct QPCV2 Ct IPC Ct QPCV2 Ct IPC Session 1 Session 2 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 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) MagVet Universal Isolation kit MagMAX Pathogen RNA/DNA Kit Oral fluids Infection at 2.00 E +03 cop pqpvc2/ ml of oral fluids Ongoing Ct QPCV2 Ct IPC Ct QPCV2 Ct IPC Session 1 Session 2 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 Extraction 5 + (35.1) + (27.0) Extraction 6 + (34.7) + (27.1) Extraction 7 + (36.3) + (27.3) Extraction 8 + (34.9) + (27.3)

26 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, EE/mL, 1:10 2 diluted in negative porcine serum #3 PCV-2 strain no 1, 1:10 3 diluted in negative porcine serum #4 PCV-2 strain no 1, 1:10 2 diluted in negative porcine serum #5 PCV-2 strain no 1, 1:10 3 diluted in negative porcine serum #6 PCV-2 strain no 1, 1:10 5 diluted in negative porcine serum #7 Negative porcine serum #8 PCV-2 strain no 1, 1:10 5 diluted in negative porcine serum 1 PCV-1 strain, 1:5 diluted ND 2 PCV-2 strain no 2, EE/mL, 1: PCV-2 strain no 1, 1: PCV-2 strain no 1, 1: PCV-2 strain no 1, 1: PCV-2 strain no 1, 1: Negative porcine serum ND 8 PCV-2 strain no 1, 1: