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1 Evaluation of the sensitivity and specificity of the AnDiaTec BoVirSL BVDV TaqMan RTPCR kit for the detection of bovine viral diarrhea virus in bovine whole blood and ear notch samples Bovine blood and ear notch samples can be tested for bovine viral diarrhea virus (BVDV) by the detection of its genomic RNA by means of the described screening assay (AnDiaTec BoVirSL BVDV TaqMan RTPCR kit). The kit provides the needed reagents for the RTPCR amplification mix, the positive and a negative control as well as the lysismix for partial lysis of ear notch samples. The amplification mix consists of the enzyme and the primersandprobesmix and is transferred into the wells of an optical microtiterplate or into optical reaction tubes. Afterwards, the RNA of a sample, positive or negative control is added. The reverse transcription (RT) of the RNA into DNA and the following PCR are done in one step. The amplification of a BVDV specific fragment is detected by the optical unit of the PCR machine due to hybridisation of fluorescencelabelled probes. The result is displayed in real time. The parallel amplification of an internal control excludes the possibility of a PCR inhibition in case of a BVDV negative result. BVDV specific fragments are detected by FAM fluorescence, the internal control is shown by VIC fluorescence. Preparation of whole blood samples. The use of pooled whole blood samples for the screening for BVD virus guarantees fast performance, high throughput and little labor. One pool consists of 100 μl whole blood from each of 10 single samples. The viral RNA is extracted from these pooled samples by means of a commercial extraction kit, either manually e.g. by the High Pure Viral RNA kit (Roche) or automated e.g. using the AGOWA mag Midi DNA Kit (on a Tecan Freedom Evo pipetting automate), which were used for this study. Cellular components are thereby lysed and redundant RNA is bound to a silica membrane. The RNA is eluted again from the membrane after several cleaning and washing steps and can be immediately used for the RT PCR. Geschäftsleitung: Dr. Johannes Kehle Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRB Persönlich haftende Gesellschafterin: AnDiaTec VerwaltungsGmbH Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRA

2 Preparation of ear notch samples. For obtaining in size and shape standardized ear notch samples, the german law 17c TierSG approved ear notch sample devices from Biopsytec (Biopsytec GmbH, Rheinbach, Germany), Typifix (Prionics AG, Zürich, Switzerland) and Allflex (Allflex, Vitre, France) were used. Partial lysis of cells containing the BVD virus was achieved using 200 μl of the readytouse lysismix and a 1h 10 min incubation at 60 C (for one hour) followed by a 10 min incubation at 95 C. Pooled ear notch homogenate supernatants were used for the screening for BVD virus, guaranteing fast performance, high throughput and little labor. One pool consists of 5 μl ear notch homogenate from each of 10 single samples. Opening of the ear notch sample tubes was performed either manually with forceps (Typifix) or with syringes (Biopsytec) or otherwise with the specific automate (Biopsytec or Typifix, respectively) the Allflex system does not require any specific instrument. Cellular components and debris proteins are highly efficient lysed and thus, the homogenate can be directly used for RTPCR. Specificity of the assay. Several reference strains of BVDV, genotype I and II, as well as other closely related or typically cattleinfecting viruses were tested to determine the specificity of the BVDV TaqMan RTPCR kit (refer to table 1). For this purpose, 1 ml of previously negatively tested whole blood was spiked with 10 3 BVD virus particles or 10 6 nonbvd virus particles. The RNA was extracted as described above and the real time RTPCR was done in triplex. All strains of BVDV were tested positive with CT values of 29 to 35. Viruses other than BVDV were not detected. Border disease virus, strain 31, which is very closely related to BVDV, was the only exception and was tested positive (refer to table 1). To confirm further the specificity of the test, 55 BVDV strains (genotype I and II type strains; some of these had been distinct BVDV strains like the recently identified BVDV strain HoBi) were tested at the German reference laboratory for BVDV (Friedrich LöfflerInstitut, Greifswald, Island of Riems, Germany)). The AnDiaTec test kit gave a correct positive result for all tested strains. Detection limit of the BVDV TaqMan RTPCR assay in whole blood samples. The aim of this study was the determination of the minimum number of virus particles detectable in 1 ml whole blood (corresponds to 10 single blood samples). Therefore, negatively tested blood was spiked with a counted number of BVDV, strain NADL (genotype Ia) or strain 890 (genotype II). The Geschäftsleitung: Dr. Johannes Kehle Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRB Persönlich haftende Gesellschafterin: AnDiaTec VerwaltungsGmbH Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRA

3 RNA was extracted as described above and was tested by the TaqMan RTPCR in triplex. The analytical detection limit was determined with 50 to 100 viral particles in 1 ml whole blood, for strain NADL as well as strain 890. This concentration of BVD virus produces CT values of 38 to 39. However, it turned out during the evaluation study that the number of virus particles present in native samples is much higher than the analytical detection limit of the RTPCR (see evaluation study). The comparison to several previously published (conventional and real time) RTPCR systems (Boye et al., 1991; Radwan et al., 1995; Hyndman et al., 1998; Letellier et al., 1999; Weinstock et al., 2001; Bhudevi et al., 2001; Mahlum et al., 2002 and Heath et al., 2003) showed a superior analytical sensitivity of the AnDiaTec test in the range between factor Reproducibility of the BVDV TaqMan RTPCR (intra assay variance). The reproducibility of the assay was tested by the following experiment: 5 positive (different CT values) and 5 negative samples were tested every 3 days with 5 different test kit lots. Table 2 shows that the assay gives highly reproducible results with an intra assay variance coefficient of less than 10 % (mean VK 0.2). Evaluation study with whole blood samples BoVirSL BVDV TaqMan RTPCR vs. commercially available ELISA and the BVDVLC LightCycler RTPCR whole blood samples were screened for further evaluation of the BoVirSL test. These samples had already been tested by commercial antigen screening assays (HerdChek BVDV Ag/Serum Plus ELISA and HerdChek BVDV Ag/Leukocytes ELISA, Idexx GmbH, Wörrstadt, Germany). Retesting of all samples was performed as blind study with the AnDiaTec BoVirSL BVDV TaqMan and the BVDVLC LightCycler RTPCR kits (22544 samples were tested blindly in the AnDiaTec laboratory (samples of 15 sources); 1625 samples were tested blind coded as part of demonstrations at 8 customers (with the existing laboratory equipment). Therefore, pools were set up consisting of 10 EDTA blood samples each, pool RNA was extracted and used for the real time RTPCR as described above. Afterwards, single blood samples forming positively tested pools were extracted in the same way and were also used for RTPCR. Table 3 summarizes the results of this experiment. All samples tested BVDV positive by ELISA and LightCycler RTPCR were approved by the BVDV TaqMan RT PCR kit. In total, 1109 samples were tested positive with all 4 methods (the BoVir test CT values of Geschäftsleitung: Dr. Johannes Kehle Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRB Persönlich haftende Gesellschafterin: AnDiaTec VerwaltungsGmbH Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRA

4 pooled samples were in the range between 17 and 26). Moreover, 27 samples (from PI animals) screened BVDV negative by ELISA were tested positive by the real time PCR tests. Nucleotide sequencing and retesting of samples from the same animals showed that these results were true positive. Further tracking revealed that 24 out of the 27 samples were samples from animals younger then 60 days (diagnostic gap as main reason for the ELISA falsenegative results). Further 44 samples negative by ELISA and positive by the real time PCR kits (CT values of pooled samples were in the range between 32 and 37) were judged as samples from transient infected animals. Serologic analysis and repeated PCR analysis from 36 animals proved this assumption. 75 samples were positive in the HerdChek BVDV Ag/Serum Plus ELISA but negative with all three other methods. These samples were validated as Serum ELISA falsepositive (according to the manufacturer, a positive sample in the HerdChek BVDV Ag/Serum Plus ELISA has to be retested in the HerdChek BVDV Leukocyte ELISA only samples positive in both ELISA kits can be judged as true positive). One sample from a known PI animal showed a negative real time RTPCR result due to inhibition (hemolytic sample). Repeated RNA extraction and subsequent RTPCR testing revealed again same results. Evaluation study with ear notch samples BoVirSL BVDV TaqMan RTPCR vs. a commercially available ELISA ear notch samples were screened for further evaluation of the BoVirSL test. These samples had already been tested by a commercial antigen screening assay (HerdChek BVDV Ag/Serum Plus ELISA, Idexx GmbH, Wörrstadt, Germany). Retesting of all samples was performed as blind study with the AnDiaTec BoVirSL BVDV TaqMan RTPCR kit (1209 samples were tested blindly in the AnDiaTec laboratory (samples of 5 sources); 219 samples were tested blind coded as part of demonstrations at 4 customers (with the existing laboratory equipment). Therefore, pools were set up consisting of 10 ear notch homogenate supernatants each, after partial lysis of the ear notch samples using the AnDiaTec lysismix as described previously. Afterwards, single homogenates forming positively tested pools were extracted in the same way and were also used for RTPCR. Table 4 summarizes the results of this experiment. In total, 833 samples were tested positive with both methods (the BoVir test CT values of pooled samples were in the range between 25 and 33). Moreover, 13 samples from previously characterized PI animals, screened BVDV negative by ELISA and were tested positive by the real Geschäftsleitung: Dr. Johannes Kehle Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRB Persönlich haftende Gesellschafterin: AnDiaTec VerwaltungsGmbH Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRA

5 time PCR tests. Additional tests using blood samples of these animals (HerdChek BVDV Ag/Serum Plus ELISA and real time PCR) showed that the RTPCR results were true positive. 91 samples were positive in the HerdChek BVDV Ag/Serum Plus ELISA but negative with our RTPCR system. Additional tests using blood samples of these animals (testing was performed with the HerdChek BVDV Ag/Serum Plus ELISA, our real time PCR and partially with an in house conventional PCR) showed that the ELISA results were falsepositive. Determination of the virus titer variance in different samples within one ear of PI animals. The variance of the virus titer in different samples (from different regions of the ear) was tested by the following experiment: in each case, 10 samples (5 taken with the Biopsytec and 5 using the Typifix system) from 15 animals were homogenized using the lysismix and tested in the real time RTPCR in triplicate. The virus titer mean variability coefficient was VK 1.5 CT. Maximal differences were within one log10 factor. Higher variability was observed when the Typifix system was used for ear notch sampling. Results of the storage experiments with ear notch homogenate supernatants. This tenability study was done with ten different samples in realtime. 5 samples were stored at 28 C and 5 at 20 C. The samples were stored under these conditions between one week and 6 months before being tested again. Table 5 summarizes the results of the study (median CT values of all samples are given). It could be shown that the quality of the sample homogenates is not affected even after 6 months of storage, if the samples are stored at 20 C. Geschäftsleitung: Dr. Johannes Kehle Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRB Persönlich haftende Gesellschafterin: AnDiaTec VerwaltungsGmbH Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRA

6 Table 1. Reference strains for the determination of BoVirSL TaqMan RTPCR kit s specificity Virus/ strain Source Reference number BVDV, strain NADL (genotype Ia) BVDV, strain New York1 (g Ib) BVDV, strain v890 (genotype II) BVDV, strain Singer (genotype Ia) BVDV, strain Oregon (g Ia) BVDV, strain 34 (genotype I) BVDV, strain 56 (genotype I) BVDV, strain A77 (genotype I) BVDV, strain 718 (genotype I) BVDV, strain 89 (genotype II) BVDV, strain V13 (genotype II) BVDV, strain V18 (genotype II) BVDV, strain 93 (genotype II) BVDV, strain 642 (genotype II) BVDV, strain A95 (genotype II) OÖ Milchprüfring, TGDLabor* OÖ Milchprüfring, TGDLabor* OÖ Milchprüfring, TGDLabor* OÖ Milchprüfring, TGDLabor* OÖ Milchprüfring, TGDLabor* University of Bern** University of Bern** OÖ Milchprüfring, TGDLabor* OÖ Milchprüfring, TGDLabor* OÖ Milchprüfring, TGDLabor* University of Bern** University of Bern** University of Bern** VR1422 VR1561 BDV, strain 31 BRSV, strain Iowa Parainfluenza type 3, strain SB Bovine rotavirus, strain C486 IBRV, strain Colorado1 Bovine herpesvirus 2, St. NY1 VR996 VR1485 VR739 VR917 VR864 VR845 * Milk survey center of Upper Austria, Laboratory for animal health service, Ried i. Innkreis, Austria ** University of Bern, Institute for human and vet. virology, Dept. of Vet. Virology, Bern, Switzerland Geschäftsleitung: Dr. Johannes Kehle Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRB Persönlich haftende Gesellschafterin: AnDiaTec VerwaltungsGmbH Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRA

7 Table 2. Reproducibility of the BoVirSL TaqMan RTPCR kit Previous result Positive Positive Positive Positive Positive CT values Test 1 Test 2 Test 3 Test 4 Test Negative Negative Negative Negative Negative Geschäftsleitung: Dr. Johannes Kehle Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRB Persönlich haftende Gesellschafterin: AnDiaTec VerwaltungsGmbH Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRA

8 Table 3. Evaluation study comparing the BoVirSL TaqMan RTPCR kit to commercial antigen ELISA kits and to the BVDVLC LightCycler RTPCR kit Number of samples Result TaqMan RTPCR Result LightCycler RTPCR Result ELISA kits 1109 positive positive positive 71 positive positive negative 1 negative negative positive * negative negative negative ** Total number of samples True Positive TaqMan RTPCR True Positive LightCycler RTPCR True Positive ELISA / / / 1181 (99.91%) (99.91%) (93.99%) * Only samples positive in both ELISA kits were counted. Sample was negative due to inhibition ** Only samples negative in both ELISA kits were counted Geschäftsleitung: Dr. Johannes Kehle Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRB Persönlich haftende Gesellschafterin: AnDiaTec VerwaltungsGmbH Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRA

9 Table 4. Evaluation study with ear notch samples comparing the BoVirSL TaqMan RTPCR kit to a commercial antigen ELISA kit Number of samples Result TaqMan RTPCR Result ELISA kits 833 positive positive 13 positive negative 91 negative positive * 363 negative negative ** Total number of samples True Positive TaqMan RTPCR True Positive ELISA / / 846 (100%) (98.46%) * falsepositive results ** truenegative results are shown Geschäftsleitung: Dr. Johannes Kehle Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRB Persönlich haftende Gesellschafterin: AnDiaTec VerwaltungsGmbH Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRA

10 Table 5. Storage experiments with ear notch samples after one week, 1 month, and 6 months (realtime) Time Day 0 Day 7 Day 30 Day 180 Date Result (CT value) Sample 1 a 27 Sample 2 a Sample 3 a Sample 4 a Sample 5 a 28 Sample 6 b Sample 7 b Sample 8 b Sample 9 b Sample 10 b a b samples stored at 28 C samples stored at 20 C Geschäftsleitung: Dr. Johannes Kehle Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRB Persönlich haftende Gesellschafterin: AnDiaTec VerwaltungsGmbH Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRA

11 Results of the tenability study The tenability study was done with three different test kit lots in realtime. Therefore, the kit components were stored under the requested conditions for 3, 6, and 12 months before being tested again. Table 1 summarizes the results of the study (median CT values of all three lots are given). It could be shown that the quality of the assay is not affected after neither 3, 6, nor 12 months of storage under the requested conditions. Table 1. Tenability study of BoVirSL TaqMan RTPCR kits after 3, 6, and 12 months (realtime) Time Day 0 Day 90 Day 180 Day 360 Date Result (CT value) Positive control a Sample 1 b Sample 2 b Sample 3 c Sample 4 b Sample 5 b Sample 6 b Sample 7 c Sample 8 b Sample 9 c Sample 10 b Negative control d a Positive control: in vitro transcribed RNA b Extracted RNA (stored at 20 C) of positive sample (from the evaluation study) c Extracted RNA (stored at 20 C) of negative sample (from the evaluation study) d Negative control: A. dest. (negative amplification control to exclude the possibility of cross contamination) Geschäftsleitung: Dr. Johannes Kehle Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRB Persönlich haftende Gesellschafterin: AnDiaTec VerwaltungsGmbH Sitz der Gesellschaft: Kornwestheim Amtsgericht Ludwigsburg HRA