Evaluation report AnDiaTec BoVir Schmallenberg-Virus real time RT-PCR Kit

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1 Evaluation report AnDiaTec BoVir Schmallenberg-Virus real time RT-PCR Kit General information. Blood (whole bood (EDTA), plasma and serum) and tissue samples from ruminants can be tested for Schmallenberg virus by the detection of its genomic RNA using the described screening assay (AnDiaTec BoVir Schmallenberg Virus real time RT-PCR kit). The kit provides the needed reagents for the RT-PCR amplification mix, the and a control. The amplification mix consists of the enzyme and the primers-and-probes-mix and is transferred into the wells of an optical microtiter plate or into optical reaction tubes. Afterwards, the RNA of a sample, or 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 Schmallenberg virus specific fragment is detected by the optical unit of the PCR machine due to hybridisation of fluorescence-labelled 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 Schmallenberg virus result. Schmallenberg virus specific fragments are detected by FAM fluorescence, the internal control is shown by VIC / HEX / JOE fluorescence. Preparation of blood and tissue samples. The viral RNA is extracted from samples by means of a commercial extraction kit, either manually (e.g. using the High Pure Viral RNA kit (Roche)) or automated (e.g. with the EasyMag RNA Kit (BioMerieux)). 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. Specificity of the assay. Extensive sequence analysis was performed in order to develop a Schmallenberg virus specific diagnostic assay. For this, the Schmallenberg virus L-, M- and S-segments of the genome were compared to all other available sequences from the genetic databases (GenBank) using the BLAST-algorithm ( Thereby, it became clear that only a few fragments from the L- and M-segment would be suitable for the development of a very specific Schmallenberg virus assay (i.p. nucleotide positions and

2 for the L-segment and nucleotide positions 950 to 1600 for the M-segment). Other genomic regions within the L- and M-segments and especially the complete S-segment are highly homologous to genomic regions of other Orthobunya viruses. These analysis led to the decision to develop an assay in one of the previously mentioned highly specific genomic regions. After some initial experiments with different primer and probe combinations a system with binding target within the M-segment was chosen. In order to evaluate the specificity of the assay, 32 previously samples (using the FLI method) from different sources were tested initially. The AnDiaTec test kit gave a correct result for all tested samples whereas other viruses (primarily RNA viruses were tested; see table 1) gave repeatedly results. Detection limit and analytical sensitivity of the BoVir Schmallenberg-Virus real time RT-PCR assay in blood samples. The aim of this study was the determination of the minimal number of viral particles detectable in 1 ml ruminant EDTA blood. Therefore, the Ct values of Schmallenberg virus samples were correlated to the results of gel electrophoresis after an end-point PCR and their comparison to a 100bp molecular marker with known copy numbers (Fermentas GmbH, St. Leon-Rot, Germany). Thus, the respective copy numbers of the samples were calculated. The RNA was extracted as described above and was tested by the real time RT-PCR in triplicate. The analytical detection limit was determined to be approximately 500 viral particles in 1 ml EDTA blood. This corresponds to a CT value of 39. Reproducibility of the BoVir Schmallenberg-Virus real time RT-PCR assay (intra assay variance). The reproducibility of the assay was tested by the following experiment: 5 samples with different viral loads and 5 s were tested every other day with 3 different test kit lots. The results show that the assay gives highly reproducible results with an intra assay variance coefficient of less than 10 % (table 2).

3 Evaluation study of the BoVir Schmallenberg-Virus real time RT-PCR kit with pretested samples (EDTA blood, serum, plasma, and tissue (mainly brain and spleen)). 124 Schmallenberg virus samples (42 EDTA blood, 21 serum, 3 plasma, and 58 tissue samples) and 110 samples (40 EDTA blood, 20 serum, 5 plasma, and 45 tissue samples) from Belgium, The Netherlands and Germany were included in the evaluation. All samples originated from cattle or small ruminants (sheep and goat), and were pre-analyzed by a real time RT-PCR developed by the Friedrich-Loeffler-Institute (Isle of Riems, Germany). The samples were provided as a blinded panel to AnDiaTec and tested by the BoVir Schmallenberg-Virus real time RT-PCR. Subsequent result assembly revealed that all 124 samples were tested clearly with the AnDiaTec real time RT-PCR (Ct values ranged from 12 to 35 and were by 3-6 Ct values lower than with the FLI-method), whereas the samples showed no amplification with this assay. A diagnostic sensitivity of 100% was achieved when using the BoVir Schmallenberg-Virus real time RT-PCR Kit (table 3). The results are summarized in table 3.

4 Table 1. Viral strains for the determination of the BoVir Schmallenberg-Virus kit s specificity Virus, strain Genotype Sample material PRRSV, Amervac Virus (Spain) PRRSV, PYRSVAC Virus (Spain) PRRSV, strain SD PRRSV, strain Lelystad EU EU NA EU BVDV, strain NADL BVDV, strain New York 1 BVDV, strain 890 Porcine Parvovirus BDV, strain 31 BRSV, strain Iowa Parainfluenza type 3, strain SB Bovine Rotavirus, strain C486 IBRV, strain Colorado-1 Bovine herpes virus 2, strain NY-1 field isolate ATCC, VR-996 ATCC, VR-1485 ATCC, VR-739 ATCC, VR-917 ATCC, VR-864 ATCC, VR-845

5 Table 2. Reproducibility of the BoVir Schmallenberg-Virus kit (intra assay variance) Inital result CT value results Day 1 Day 2 Day

6 Table 3. Evaluation study of the BoVir Schmallenberg-Virus real time RT-PCR Kit Number of samples Result BoVir RT-PCR Pre-test result Total number of samples Total BoVir RT-PCR Total Pre-test True BoVir RT-PCR True Pre-test Diagnostic sensitivity 100% 124 /