September Validation Report. virotype ASFV PCR Kit. For the detection of DNA from African swine fever virus (ASFV)

Transcription

1 September 2015 Validation Report virotype ASFV PCR Kit For the detection of DNA from African swine fever virus (ASFV)

2 1 Introduction 1.1 Intended Use The virotype ASFV PCR Kit is intended for the detection of DNA from African swine fever virus (ASFV) in serum, plasma, EDTA-blood, tissue, and swab samples from pigs and wild boar. The kit is approved by the Friedrich-Loeffler-Institut and licensed in accordance with 11 (2) of the German Animal Health Act (FLI-B 670) for use in Germany for veterinary diagnostic procedures. For veterinary use only. 1.2 General Information The virotype ASFV PCR Kit is a highly sensitive and specific solution for the detection of DNA from African swine fever virus (ASFV) in samples from pigs and wild boar. African swine fever (ASF) is one of the most important infectious viral diseases of swine of all ages and causes a wide range of clinical signs characterized by a high rate of morbidity and mortality. The disease is notifiable to the World Organization for Animal Health (OIE). The causative agent is a double-stranded DNA virus belonging to the family Asfarviridae, genus Asfivirus. ASF virus can be transmitted by vectors (soft ticks of the genus Ornithodoros) therefore classified as Arbovirus (arthropod-borne virus). The high sensitivity of the virotype ASFV PCR Kit allows the early detection of the pathogen in individual as well as in pooled samples. 1.3 Description of the Test Principle Polymerase chain reaction (PCR) is based on the amplification of specific regions of the pathogen genome. In real-time PCR, the amplified product is detected using fluorescent dyes. These are usually linked to oligonucleotide probes that bind specifically to the amplified product. Monitoring the fluorescence intensities during the PCR run (i.e., in real time) allows the detection of the accumulating product without the need to re-open the reaction tubes afterward. The virotype ASFV PCR Kit contains all of the necessary reagents for the detection of ASFV DNA, including a and negative control. An internal control excludes the possibility of false-negative results. The kit uses two specific primer/probe combinations: one for ASFV DNA yielding FAM fluorescence and one for a housekeeping gene (β-actin DNA) present within the sample yielding HEX fluorescence. A Positive Control serves to verify the functionality of the reaction mix for the amplification of the ASFV DNA target. virotype ASFV PCR Kit Validation Report 2

3 1.4 Kit Contents virotype ASFV PCR Kit (24) (96) Catalog no Number of reactions Master Mix (tube with orange cap) includes enzymes, primers, and probes 1 x 500 µl 2 x 980 µl Positive Control (tube with red cap) 1 x 25 µl 1 x 70 µl Negative Control (tube with blue cap) 1x 25 µl 1 x 70 µl Handbook Storage The components of the virotype ASFV PCR Kit should be stored at -30 C to -15 C and are stable until the expiration date stated on the label. Avoid repeated thawing and freezing (>2x), as this may reduce assay sensitivity. Freeze the components in aliquots if they will only be used intermittently. 1.6 Equipment and Reagents to Be Supplied by User When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier. Pipets Nuclease-free aerosol-resistant pipet tips with filters Sterile 1.5 ml Eppendorf tubes Nuclease-free (RNase/DNase-free) consumables. Special care should be taken to avoid nuclease contamination of all reagents and consumables used to set up PCR for sensitive identification of viral nucleic acids. Benchtop centrifuge with rotor for 1.5 ml tubes Cooling device or ice Rotor-Gene Q or 96-well plate real-time cycler with appropriate fluorescent channels Rotor-Gene Q software version or higher, or appropriate software for chosen 96-well plate cycler virotype ASFV PCR Kit Validation Report 3

4 Strip Tubes and Caps, 0.1 ml, for use with Rotor-Gene Q (cat. no or ) or 96-well optical microplate with optical sealing film or cover for chosen 96-well plate real-time cycler 1.7 DNA extraction The virotype ASFV RT-PCR Kit can be used for the detection of ASFV DNA from serum, plasma, EDTA-blood, tissue and swab samples from swine. Due to the high sensitivity of the test individual or pooled samples can be tested. Pools up to 20 individual serum, plasma, EDTA-blood or tissue samples can be used, provided that the sample quality is good. It is recommended to test dead wildlife samples on an individual basis. Prior to real-time PCR, viral DNA must be extracted from the starting material. QIAGEN offers a range of products for DNA extraction from animal samples. DNA extraction using kits based on spin-column technology can be automated using the QIAcube. For an overview of validated extraction kits and methods see Table 1. If real-time PCR is not performed immediately after extraction, store the DNA at -20 C or at 70 C for longer storage. Table 1. Overview of recommended extraction kits. Sample type whole blood swabs tissue Extraction kit QIAamp Viral RNA Mini Kit QIAamp cador Pathogen Mini Kit QIAamp DNA Mini Kit DNeasy Blood & Tissue Kit QIAamp Viral RNA Mini Kit QIAamp cador Pathogen Mini Kit QIAamp DNA Mini Kit QIAamp cador Pathogen Mini Kit DNeasy Blood & Tissue Kit QIAamp DNA Mini Kit Kit protocol Manual extraction method Purification of Viral RNA (Spin Protocol)* Purification of Pathogen Nucleic Acids from Fluid Samples DNA Purification from Blood or Body Fluids (Spin Protocol) Purification of Total DNA from Animal Blood or Cells (Spin-Column Protocol) Purification of Viral RNA (Spin Protocol)* Purification of Pathogen Nucleic Acids from Fluid Samples DNA Purification from Buccal Swabs Pretreatment T1 - Mechanical Disruption of Tissue Purification of Total DNA from Animal Blood or Cells (Spin-Column Protocol) DNA Purification from Tissues virotype ASFV PCR Kit Validation Report 4

5 oral fluid whole blood swabs tissue oral fluid QIAamp Viral RNA Mini Kit QIAamp cador Pathogen Mini Kit QIAamp DNA Mini Kit DNeasy Blood & Tissue Kit QIAamp cador Pathogen Mini Kit QIAamp DNA Mini Kit DNeasy Blood & Tissue Kit QIAamp Viral RNA Mini Kit QIAamp cador Pathogen Mini Kit QIAamp DNA Mini Kit QIAamp cador Pathogen Mini Kit DNeasy Blood & Tissue Kit QIAamp cador Pathogen Mini Kit QIAamp DNA Mini Kit Purification of Viral RNA (Spin Protocol)* Purification of Pathogen Nucleic Acids from Fluid Samples DNA Purification from Blood or Body Fluids (Spin Protocol) Purification of Total DNA from Animal Blood or Cells (Spin-Column Protocol) Automated extraction method Virus_QIAampcadorPathogenMini_Fluids_Standard_V2 (QC ) DNA_QIAampDNABloodMini_BloodOrBodyFluid_ManualLysis_V1 (QC ) DNA_DNA_DNeasyBloodAndTissue_AnimalBoodOrCells_Standard_V2 (QC ) QC - Virus_QIAampViralRNA_BodyFluid_Standard_V2 (QC )* Virus_QIAampcadorPathogenMini_Fluids_Standard_V2 (QC ) DNA_QIAampDNABloodMini_BuccalSwab_ BuccalSwabLysisAndPurification_V3 (QC ) Virus_QIAampcadorPathogenMini_Fluids_Standard_V2 (QC ) DNA_DNeasyBloodAndTissue_TissuesAndRodentTails_Standard_V1 (QC ) Virus_QIAampcadorPathogenMini_Fluids_Standard_V2 (QC ) DNA_QIAampDNABloodMini_BloodOrBodyFluid_ManualLysis_V1 (QC ) DNeasy Blood & DNA_DNA_DNeasyBloodAndTissue_AnimalBoodOrCells_Standard_V2 Tissue Kit (QC ) (* allows simultaneous extraction of ASFV DNA and CSFV RNA; QC = QIAcube) virotype ASFV PCR Kit Validation Report 5

6 2 Procedure Important points before starting Include at least one control (Positive Control) and one negative control (Negative Control) per PCR run. Before beginning the procedure, read through the protocol and ensure that you are familiar with the operation of the chosen real-time PCR cycler. Perform the protocol without interruption. Things to do before starting Thaw all reagents on ice and protect from light. Maintain reagents on ice during PCR setup. Before use, spin the reagents briefly Procedure 1. Pipet 20 µl of the Master Mix into each reaction tube. Then add 5 µl of the sample DNA (Table 2). Include and negative control reactions. Positive control: Use 5 µl of the control (Positive Control) instead of sample DNA. Negative control: Use 5 µl of the negative control (Negative Control) instead of sample DNA. Table 2. Preparation of reaction mix Component Volume Master mix 20 µl Sample 5 µl Total volume 25 µl 2. Close the reaction tubes with the corresponding caps. 3. Set the filters for the reporter dyes in the software of your thermal cycler according to Table 3. Select the green and yellow channels on the Rotor-Gene Q. Important: Set a fixed gain of +4 in the green and +1 in the yellow channels to ensure optimal fluorescence gains for the Pathogen and the Internal Control assays. virotype ASFV PCR Kit Validation Report 6

7 Table 3. Filter settings for reporter Pathogen/ IC Reporter ASFV/unknown sample 96-well plate FAM Rotor-Gene Q green Internal control HEX/JOE * yellow Passive reference ROX - * Use the option appropriate for your thermal cycler. Internal reference for use with the Applied Biosystems ABI PRISM Sequence Detection Systems. 4. Run the real-time PCR protocol according to Table 4. Table 4. Real-time PCR protocol for ASFV Temperature Time Number of cycles 95 C 15 min 1 95 C 15 s 60 C 60 s 40 Fluorescence data collection. Approximate run time (Rotor-Gene Q): 96 min 5. Run the real-time RT-PCR protocol according to Table 5 if running the virotype CSFV assay simultaneously. Table 5. Real-time RT-PCR protocol for simultaneous amplification of ASFV and CSFV 1 Temperature Time Number of cycles 45 C 10 min 1 95 C 10 min 1 95 C 15 s 57 C 30 s C 35 s 1 valid for virotype CSFV RT-PCR assay only Fluorescence data collection. Approximate run time (Rotor-Gene Q): 118min virotype ASFV PCR Kit Validation Report 7

8 3 Data Interpretation Interpretation of results For the assay to be valid the Positive Control must give a signal in both the FAM and HEX channels with a C T * <35. The Negative Control must give no signal. The following results are possible if working with unknown samples. The possible sample results are also summarized in Table 6. The sample is for ASFV, and the assay is valid, if the following criteria are met: The sample yields a signal in both the FAM and HEX channel. The Positive Control yields a signal in both the FAM and HEX channel. The Negative Control does not yield a signal in the FAM and HEX channel. Note that very high concentrations of ASFV DNA in the sample may lead to a reduced HEX signal or no HEX signal due to competition with the internal control. 1 The sample is negative for ASFV, and the assay is valid, if the following criteria are met: The sample yields a signal in the HEX channel but not in the FAM channel. The Positive Control yields a signal in both the FAM and HEX channel. The Negative Control does not yield a signal in the FAM and HEX channel. A HEX signal means that extraction and amplification were successful as the housekeeping gene within the sample is amplified. However, if the C T value of the internal control is >35, pooled or individual samples could be partially inhibited. In such cases the respective individual samples should be diluted (e.g., diluted 1:5) in nuclease free water and retested. The sample results are inconclusive, and the assay is invalid, if the following occurs: The sample yields no signal in the FAM and HEX channel. If no signal is detected in both the FAM (pathogen) and the HEX (Internal Control, IC) channel, the result is inconclusive. The absence of a signal for the Internal Control indicates PCR inhibition and/or other malfunctions. To check for inhibition, we recommend 1:5 dilution of the sample DNA in nuclease free water, to repeat the DNA extraction, or repeat the whole test procedure starting with new sample material. Check that there is a fluorescence signal in the FAM channel for the control reaction (Positive Control). Absence of a signal for the Positive Control indicates an error, which could be due to incorrect setup of the master mix or incorrect cycling conditions. * Threshold cycle (C T ) cycle at which the amplification plot crosses the threshold, i.e., there is the first clearly detectable increase in fluorescence. Green and yellow on the Rotor-Gene Q. virotype ASFV PCR Kit Validation Report 8

9 Table 6. Results interpretation table* Sample result Reporter FAM (pathogen) ASFV X X ASFV (strong ) ASFV negative Inconclusive result HEX (IC) * Interpretation of sample results can be determined provided and negative control reactions are performed. The control must yield a signal in both the FAM and HEX channels. The negative control must yield no signal in the FAM and HEX channels. For a complete explanation of possible sample results please refer to Data Analysis and Interpretation. X X virotype ASFV PCR Kit Validation Report 9

10 4 Characteristics of the Test 4.1 Analytical Sensitivity of virotype ASFV PCR Kit Using the ASFV PCR Program The high analytical sensitivity of the virotype ASFV PCR Kit was shown by testing titration series of ASFV in vitro DNA [10 7 to 10-1 copies/well]. The testing was performed in triplicates of ten-fold dilutions, using the following real-time PCR protocol: 15 min 95 C, 40 cycles: 15 sec 95 C, 60 sec 60 C. The results are shown in Table 7 and Figure 1. Table 7: Analytical Sensitivity of virotype ASFV using the virotype ASFV PCR program (Rotor-Gene Q). Number of ASFV (FAM) Replicates copies C T C T mean sd Result No C T negative No C T No C T - negative 8 No C T negative 9 No C T negative No C T No C T - negative 9 No C T negative virotype ASFV PCR Kit Validation Report 10

11 ASFV-DNA copies/well correlation coefficient R 2 = slope efficiency 98% Figure 1: Titration series and standard curve of ASFV in vitro DNA using virotype ASFV. Result: Using virotype ASFV a high correlation between the number of copies and the amount of amplified product was demonstrated. The assay allows the detection of down to 10 copies of ASFV-DNA (Table 7, Figure 1; correlation coefficient of with an efficiency of 98%). virotype ASFV PCR Kit Validation Report 11

12 4.1.2 Using the virotype CSFV RT-PCR Program Alternatively, the virotype CSFV RT-PCR program (45 C 10 min, 95 C 10 min, 40 cycles: 95 C 15 sec, 57 C 30 sec, 72 C 35 sec) can be used if the test is performed simultaneously with the virotype CSFV assay in the same real-time cycler. The analytical sensitivity of the virotype ASFV was tested using the alternative RT-PCR program. Titration series of ASFV in vitro DNA [10 7 to 10-1 copies/well] were used in triplicates of ten-fold dilutions to test the performance. The results are shown in Tables 8 and Figures 2. Table 8: Analytical Sensitivity of virotype ASFV using the virotype CSFV RT-PCR program (Rotor-Gene Q). Copy ASFV (FAM) Replicates Number C T C T mean sd Result No C T - negative No C T No C T negative 8 No C T negative 9 No C T - negative No C T No C T - negative 9 No C T negative virotype ASFV PCR Kit Validation Report 12

13 ASFV-DNA copies/well correlation coefficient R 2 = slope efficiency 101% Figure 2: Titration series and standard curve of ASFV in vitro DNA using the virotype CSFV RT-PCR program. Result: Using the virotype CSFV RT-PCR program and the virotype ASFV PCR assay, a high correlation between the number of copies and the amount of amplified product was demonstrated for ASFV-DNA (R 2 = 0.999, Efficiency = 101%). It is possible to detect 10 copies of ASFV-DNA (Table 8, Figure 2). virotype ASFV PCR Kit Validation Report 13

14 Conclusion The analytical sensitivity for both real-time PCR programs (virotype ASFV PCR program and virotype CSFV RT-PCR program) is highly similar. Therefore, the virotype CSFV RT-PCR protocol is a good alternative if ASFV and CSFV assays are tested simultaneously in the same thermal cycler. 4.2 Analytical Specificity of virotype ASFV PCR Kit ASFV is a double-stranded DNA virus of the family Asfarviridae. There are 22 genotypes identified world-wide, with genotypes I and II being most prevalent in Europe to date. The ASFV-specific amplification is combined with an amplification of β-actin DNA as a duplex- PCR using the same sample. Primer and probe detecting ASFV bind to the ASFV p72 gene (King et al.). The amplification of β-actin is at the same time an extraction control, as it will only be amplified after successful extraction. The amplification of β-actin does not interfere with the ASFV signal. Only very high concentrations of ASFV DNA could cause a failure of the β-actin signal due to competition during the PCR. 4.3 Cross-reactivity to other relevant porcine pathogens for differential diagnosis Cross-reactivity was tested using samples either for Classical Swine Fever Virus (CSFV), Porcine Reproductive Respiratory Syndrome Virus (PRRSV), Influenza A Virus, and porcine circovirus 2 (pcv-2). The samples were kindly provided by the FLI and different State Diagnostic Laboratories (Bavaria, Rhineland-Palatinate, North Rhine-Westphalia; Table 9). Table 9: Testing cross-reactivity of virotype ASFV to other porcine viral pathogens. Pathogen Tested number of samples virotype ASFV PCR Kit Reference Test true negative false CSFV PRRSV Influenza A pcv Result: No cross-reactivity to other relevant porcine viral pathogens was detected using virotype ASFV. Conclusion virotype ASFV specifically detects ASFV DNA. virotype ASFV PCR Kit Validation Report 14

15 4.4 Diagnostic Sensitivity and Specificity Diagnostic Sensitivity and Specificity Diagnostic sensitivity measures the proportion of actual s which are correctly identified as such. Actual s scoring a negative result are considered false-negative. Diagnostic sensitivity = (true-/[true- + false-negative])*100 Diagnostic specificity measures the proportion of actual negatives which are correctly identified as such. Actual negatives scoring a result are considered false-. Diagnostic specificity = (true-negative/[true-negative + false-])*100 For validation of virotype ASFV, 317 samples (blood, serum, tissue, faecal and oropharyngeal swabs) were tested. Reference samples (113 ASFV-, 16 ASFV-negative) were kindly provided by the Friedrich-Loeffler-Institut (FLI). The ASFV- sample panel comprised of three different genotypes (Sardinia, Armenia and Kenia) corresponding to genotype I, II and X, respectively. Furthermore, 188 ASFV-negative samples were provided by the FLI and different State Diagnostic Laboratories (Bavaria, Rhineland-Palatinate, and North Rhine-Westphalia). All samples were extracted using either the QIAamp Viral RNA Mini Kit or the QIAamp cador Pathogen Mini Kit (QIAGEN) following the manufacturer s instructions and tested using the virotype ASFV PCR Kit. The results are shown in Table 10. Table 10: Diagnostic sensitivity, specificity and efficiency of the virotype ASFV PCR Kit virotype ASFV PCR Kit Reference Test Total number of samples 317 Reference 113 Reference negative 204 ASFV- 112 true- 112 false- 0 ASFV-negative 205 False-negative 1 true- negative 204 Diagnostic sensitivity % Diagnostic specificity % Diagnostic efficiency % Result: Testing this panel of samples resulted in a diagnostic sensitivity of 99.12% and a diagnostic specificity of 100%. The resulting diagnostic efficiency is 99.68%. virotype ASFV PCR Kit Validation Report 15

16 4.4.2 Testing of wild boar and pig samples - a comparison between FLI in-house PCR and virotype ASFV (virotype ASFV PCR and virotype CSFV RT-PCR program) ASFV- DNA samples from experimentally infected wild boar and domestic pigs were tested running two different PCR programs (ASFV-specific and alternative CSFV-PCR program) using the virotype ASFV PCR Kit. The results are compared to the FLI PCR results. All samples were kindly provided by the FLI. The results are presented in Tables 11 (wild boar) and 12 (domestic pigs). Table 11: Validation results of ASFV- samples from experimentally infected wild boar. Samples were tested using both virotype ASFV-specific and virotype CSFV RT-PCR program (Rotor-Gene Q). Wild boar AFSV strain Armenia Sardinia Animal ID ws01 ws07 ws11 ws14 ws12 virotype virotype ASFV program CSFV program DPI* Sample type FLI C T FAM C T HEX C T FAM C T HEX 14 oropharyngeal swab oropharyngeal swab lymph node spleen tonsil blood blood blood blood lymph node spleen tonsil oropharyngeal swab oropharyngeal swab oropharyngeal swab blood blood blood blood lymph node spleen tonsil blood blood lymph node tonsil lung bone marrow salivary glond oropharyngeal swab virotype ASFV PCR Kit Validation Report 16

17 8 oropharyngeal swab Sardinia ws12 4 faecal swab faecal swab * DPI = days post infection Table 12: Validation results of ASFV- samples from experimentally infected domestic pigs. Samples were tested using both virotype ASFV-specific and virotype CSFV RT-PCR program (Rotor-Gene Q). Domestic pig AFSV strain Kenia05 Armenia Animal ID hs07 hs17 hs24 virotype virotype ASFV program CSFV program DPI* Sample type FLI C T FAM C T HEX C T FAM C T HEX 7 faecal swab faecal swab blood lymph node bone marrow tonsil salivary gland lung blood blood lymph node spleen tonsil blood blood oropharyngeal swab oropharyngeal swab oropharyngeal swab lymph node spleen tonsil * DPI = days post infection Result: All tested ASFV- DNA samples of experimentally infected wild boar and domestic pigs scored correctly using the virotype ASFV PCR Kit. The alternative virotype CSFV RT-PCR program resulted in highly similar C T values as were obtained using the ASFV-specific PCR program. Conclusion If testing porcine samples both for ASFV DNA and CSFV RNA using virotype assays, both can be performed simultaneously in one thermal cycler using the virotype CSFV RT-PCR program. virotype ASFV PCR Kit Validation Report 17

18 4.5 Testing of pooled samples Pools were generated by diluting selected ASFV- DNA samples (FLI reference samples) in appropriate ASFV-negative porcine DNA from blood (extracted using the QIAamp Viral RNA Mini Kit [QIAGEN] following manufacturer s instructions). Using virotype ASFV, pathogen and Internal Control signals could be detected in all samples (Table 13). Table 13: Testing of pooled oral fluid samples. Pool Sample (ASFV, C T FAM) size single Pool size Sample (IC, C T HEX) single Result: Using the selected reference samples provided by the FLI, both ASFV (FAM) as well as Internal Control (HEX) signals were detected in pools sizes up to 20. virotype ASFV PCR Kit Validation Report 18

19 4.6 Reproducibility Intra-assay Variance Three ASFV- and one ASFV-negative in vitro DNA sample, and Positive and Negative Control (PC, NC) of the test kit were tested in a six-fold setup in the same PCR run analysing the intra-assay variance of virotype ASFV. The mean value (MV), standard deviation (sd) and coefficient of variation (CV) were calculated (Table 14). Table 14: Intra-assay variance of ASFV (FAM) and Internal Control (HEX). Presented are C T values for four DNA samples (3, 1 negative), Positive and Negative Control (PC, NC) in one PCR run (six-fold; Rotor-Gene Q). ASFV (FAM signal) Run PC Neg NC No C T No C T No C T No C T No C T No C T No C T No C T No C T No C T No C T No C T MV sd CV Internal Control (HEX signal) Run PC Neg NC No C T No C T No C T No C T No C T No C T MV sd CV Result: For the intra-assay variance of the ASFV-specific FAM signal coefficients of variation were calculated 0.34% to 1.15%. For the intra-assay variance of the Internal Control-specific HEX signal coefficients of variation were calculated 0.50 to 1.83 % Inter-assay Variance Six ASFV- and one ASFV-negative in vitro DNA sample, and Positive and Negative Control (PC, NC) of the test kit were tested in seven independent PCR runs analysing the interassay variance of virotype ASFV. The mean value (MV), standard deviation (sd) and coefficient of variation (CV) were calculated (Table 15). virotype ASFV PCR Kit Validation Report 19

20 Table 15: Inter-assay variance of ASFV (FAM) and Internal Control (HEX). Presented are C T values for seven DNA samples (6, 1 negative), Positive and Negative Control (PC, NC) in seven independent PCR run (Rotor- Gene Q). ASFV (FAM signal) Run PC Neg NC No C T No C T No C T No C T No C T No C T No C T No C T No C T No C T No C T No C T No C T No C T MV sd CV Internal Control (HEX signal) Run PC Neg NC No C T No C T No C T No C T No C T No C T No C T MV sd CV Result: For the inter-assay variance of the ASFV-specific FAM signal coefficients of variation were calculated 0.36% to 1.30%. For the intra-assay variance of the Internal Control-specific HEX signal coefficients of variation were calculated 0.34 to 1.21 % Batch-to-Batch Variance The Positive Control of the test kit was tested in a six-fold setup using two different batches of the virotype ASFV PCR Kit (ASF , ASF ). The mean value (MV), standard deviation (sd) and coefficient of variation (CV) were calculated (Table 16). virotype ASFV PCR Kit Validation Report 20

21 Table 16: Batch-to-Batch variance for virotype ASFV PCR Kit (presented are C T values for the PC in a six-fold setup using two batches). ASFV (FAM signal) Sample ASF ASF PC_ PC_ PC_ PC_ PC_ PC_ MV sd CV Internal Control (HEX signal) Sample ASF ASF PC_ PC_ PC_ PC_ PC_ PC_ MV sd CV Additionally, a titration series of ASFV in vitro DNA was tested using these two different batches of virotype ASFV. The difference in C T -values is shown in Table 17. Table 17: Batch-to-Batch variance for virotype ASFV PCR Kit (presented are difference in C T values for an in vitro DNA titration series using two batches) ASFV (FAM signal) Sample ASF ASF C T 1x x x x x x x Result: The variation of coefficient of the FAM and HEX signals of the Positive Control were calculated 0.20% to 0.72% (Table 16). The C T values obtained for the titration series of in vitro DNA show only minimal deviations between both tested batches (Table 17). virotype ASFV PCR Kit Validation Report 21

22 Conclusion The results obtained for intra-assay variance, inter-assay variance and batch-to-batch variance show an excellent reproducibility using the virotype ASFV PCR Kit. 4.7 Stability of the virotype ASFV PCR Kit Validating the stability of the virotype ASFV PCR Kit, a panel of titrated in vitro ASFV DNA was tested using two different batches (ASF , ASF ) at the time of production and after four and three months storage, respectively. The results are summarized in Table 18 (ASF ) and Table 19 (ASF ). Table 18: Stability of virotype ASFV PCR Kit (ASF ) ASF C T FAM Aug 2014 Dec 2014 C Tmean C Tmean C T 1x x x x x x x Table 19: Stability of virotype ASFV PCR Kit (ASF ) ASF C T FAM Sep 2014 Dec 2014 C Tmean C Tmean C T 1x x x x x x x Result: After four months storage, both batches show only minimal deviations in their C T values ( C T for ASF ; C T for ASF ). If the test kit is stored according to the instructions (protected from light, at -30 C to -15 C), and freeze-thawed not more often than two times, a shelf life of 18 months can be assumed. virotype ASFV PCR Kit Validation Report 22

23 4.8 External evaluation of the virotype ASFV PCR Kit The virotype ASFV PCR Kit was externally evaluated by testing ASFV isolates representing 20 different p72 genotypes. The results were shown in Table 20 in comparison to the OIE-PCR (OIE 2012). Table 20: Comparison of the virotype ASFV PCR Kit with the OIE-PCR testing different ASFV genotypes. ASFV (C T FAM) ASFV isolate GENOTYPE virotype OIE-PCR AFSV PCR Kit E75 I Maur08/1 II RSA2008/1 III RSA/W/1/99 IV Moz64 V SPEC/265 VI RSA/03/7 VII MwLil20/1 VIII Ken06.Bus IX BUR90/1 X KAB6/2 XI MZI92/1 XII SUM14/11 XIII NYA1/2 XIV TAN/2008/1 XV TAN2003/2 XVI NAM/P/1/95 XVII XX SPEC53 XXI RSA2008/2 XXII Result: 20 different ASFV-genotypes (of the 22 ASFV-genotypes currently known) that are detected by the OIE Reference-PCR were also detected by the virotype ASFV PCR Kit (Table 20). virotype ASFV PCR Kit Validation Report 23

24 Australia Austria Belgium Brazil Canada China Denmark Finland France Germany Hong Kong India Ireland Italy Japan Korea (South) Luxembourg Mexico The Netherlands Norway Singapore Sweden Switzerland UK USA