V8_2011-01-14 VIROTYPE BVDV Instructions for Use Real-Time RT-PCR Test Kit for Detection of Bovine Viral Diarrhoea Virus In vitro Diagnostic Kit for Veterinary Medicine Registered in Accordance with 17c of the German Law on Animal Diseases Registration No.: FLI-B 451 VIROTYPE BVDV 25 reactions Cat. No. 05-101/25 VIROTYPE BVDV 96 reactions Cat. No. 05-101/96 VIROTYPE BVDV 480 reactions Cat. No. 05-101/480 Applications The product group VIROTYPE comprises test systems for the identification of viral pathogens by real-time PCR. VIROTYPE BVDV is a real-time RT-PCR test kit for the detection of the virus causing Bovine Viral Diarrhoea and Mucosal Disease. Blood, plasma, serum, milk and ear tissue samples from cattle can be tested (individual or pooled samples). Production & Support: LDL - Labor Diagnostik GmbH Leipzig Deutscher Platz 5b I 04103 Leipzig I Germany Phone +49 (0)341/12454 0 I Fax +49 (0)341/12454 60 info@lab-leipzig.de I www.lab-leipzig.de
- 2 General Information Bovine Viral Diarrhoea (BVD) and Mucosal Disease (MD) are caused by Bovine Viral Diarrhoea Virus (BVDV I, II), a single-stranded RNA virus. It is grouped into the genus of pestiviruses, as is Classical Swine Fever Virus and Border Disease Virus in sheep. BVD is an economically important infectious disease of cattle. Depending on the immune status of the animals, BVDV infections may lead to gastro-intestinal and respiratory symptoms of different severity as well as to reproductive problems. These are caused by transplacental infection of the foetus leading to abortions, congenital malformations, and in case of infection before immunocompetence to persistently infected (PI or viraemic) calves. PI animals only emerge through prenatal infection, whereas postnatal infections lead to transient viraemia, inducing the production of neutralising antibodies. Unidentified PI animals are responsible for the spread of BVDV as they excrete high doses of the virus during their whole life. They may thus infect pregnant animals which in turn may give birth to new PI animals. The major aim to successfully combat the disease is the early identification of those PI animals. The high sensitivity of VIROTYPE BVDV ensures the early detection of the pathogen in individual as well as in pooled samples (pool size up to 50 individual samples for blood, plasma, serum, pool size up to 100 individual samples for milk or up to 25 individual ear tissue samples). Description of the Test Principle VIROTYPE BVDV includes all reagents for the detection of BVDV RNA, as well as positive and negative control. Reverse transcription (RT) of the viral RNA and subsequent amplification of the cdna by Polymerase Chain Reaction (PCR) are performed in one tube; this reduces the risk of contaminations to a minimum. The reporters of the probes will emit fluorescence in proportion to the amount of amplificate produced. Thus, the reaction can be monitored in real-time (real-time PCR). False negative results are excluded by using an internal control. VIROTYPE BVDV uses two specific combinations of primer with probe, one for pestiviruses giving FAM fluorescence and one for the internal control giving HEX fluorescence.
- 3 Content 25 Reactions 96 Reactions 480 Reactions PCR Mix (yellow cap) includes primers, probes, and internal control 560 µl 2 x 1 ml 6 x 1,65 ml Enzyme Mix (green cap) 7,5 µl 26 µl 2 x 65 µl Positive Control (red cap) 25 µl 50 µl 2 x 50 µl Negative Control (blue cap) 25 µl 50 µl 1 x 100 µl Storage and Shelf Life Immediately upon receipt store the kit at -20 C, protected from light. Under these conditions the kit is stable until the indicated expiration date. Do not use the reagents past expiration date. Repeated freezing and thawing (> 4 cycles) must be avoided. Additional Material and Equipment Required - RNA extraction kit - Pipettes (0.5 µl 1000 µl) - Sterile filter tips (aerosol resistent) - Sterile 1.5 ml Eppendorf tubes - Bench-top centrifuge - Optical 96-well reaction plate or reaction tubes - Optical sealing film or covers - Real-time PCR thermal cycler (Stratagene Mx3000P, Stratagene Mx3005P, ABI PRISM, or similar) Trademarks and Patents VIROTYPE IS A REGISTERED TRADEMARK. PATENT RIGHTS PROTECT PCR. THE PURCHASE OF THIS PRODUCT GRANTS THE PURCHASER RIGHTS UNDER CERTAIN ROCHE PATENTS TO USE IT FOR PROVIDING VETERINARY IN VITRO DIAGNOSTIC TESTING. NO GENERAL PATENT OR OTHER LICENSE OF ANY KIND OTHER THAN THIS SPECIFIC RIGHT OF USE FROM PURCHASE IS GRANTED HEREBY.
- 4 Precautions and Warnings The test should only be performed by persons qualified for laboratory work. The components of the test kit may not be contaminated or mixed with components from other batches. Sample preparation and amplification should be carried out in separate rooms. Take appropriate safety measures for working in laboratories and stick to GLP rules. Wear gloves all the time. The PCR Mix contains < 0.1 % sodium azide (NaN3). All sample residues and objects which have come into contact with samples must be decontaminated or disposed of as potentially infective material. Please stick rigorously to the BVDV real-time RT-PCR protocol (page 5). For veterinary use only. Sample Material VIROTYPE BVDV is suitable for the detection of BVDV RNA from bovine blood, plasma, serum, milk and ear tissue samples. Preferably use EDTA plasma because viral RNA is minimally degraded in the presence of EDTA. Due to the high sensitivity of the test, pools consisting of 50 individual blood samples (blood, plasma, serum), pools of 100 individual milk samples and pooled ear tissue samples consisting of 25 individual samples may be analysed. However, the optimum pool size depends on the regional prevalence for BVDV and on the age of the animals. RNA Extraction For the isolation of BVDV RNA we recommend the following kit systems: QIAamp Viral RNA Mini (QIAGEN) QIAamp MinElute Virus Spin (QIAGEN) NucleoSpin RNA Virus (Macherey-Nagel) MagMAX Viral RNA Isolation Kit (AM 1836, Applied Biosystems) RNeasy Mini Kit (QIAGEN), user-developed protocol RNeasy Fibrous Tissue Mini (QIAGEN) Plasma, Serum, EDTA-blood, Milk Plasma, Serum, EDTA-blood Plasma, Serum, EDTA-blood Plasma, Serum Milk Ear tissue Store the isolated RNA at -70 C (-20 C or below) in case the real-time RT-PCR is not performed immediately after RNA extraction.
- 5 For rapid preparation of ear tissue samples (diameter 2-3 mm) without RNA isolation LDL recommends VIROTYPE TLR. Ear tissue lysates should be stored at -20 C or at 2-8 C for up to 12 h. BVDV Real-Time RT-PCR Protocol Please read the entire protocol before starting the test procedure. RNA is unstable, please perform this protocol uninterrupted. The Enzyme Mix should be taken out of the refrigerator at -20 C immediately before use, always kept on ice, and returned to -20 C immediately after use. 1. Before use, spin the reagents shortly. Perform all pipetting steps on ice. 2. The master mix is prepared by mixing the PCR Mix (yellow cap) with the Enzyme Mix (green cap). Pipetting scheme: No. of reactions 1 25 96 480 PCR Mix (yellow cap) 19,75 µl 493,75 µl 1.896,00 µl 9.480,00 µl Enzyme Mix (green cap) 0,25 µl 6,25 µl 24,00 µl 120,00 µl master mix volume 20,00 µl 500,00 µl 1.920,00 µl 9.600,00 µl Calculate the volume of the master mix for the number of the reactions (samples + controls) plus a reserve. Perform at least one Positive and one Negative Control for each run. Pipet the PCR Mix and the Enzyme Mix in a sterile reaction tube, mix, spin shortly. 3. Transfer 20 µl of the master mix into each well of the optical micro titre plate or into optical reaction tubes. 4. Add 5 µl of the isolated RNA or of controls (red and blue) and mix. Thus, the final volume of a test is 25 µl. Immediately cover the wells with sealing film, or close caps, respectively. Spin shortly if necessary.
- 6 Use the following settings for reporter and quencher in the software of your thermal cycler: Reporter Quencher BVDV FAM TAMRA Internal control HEX/JOE * TAMRA Passive reference ROX * use option available in your thermal cycler Real-time RT-PCR protocol: 50 C 20 min 95 C 15 min 95 C 30 s 40 cycles 57 C 45 s Measure at the end of this step 68 C 45 s Test Validation For valid results, the positive control 1 must have a FAM signal and a HEX signal of a Ct-value 2 less than 36 (Ct < 36). The negative control does not have a FAM signal but a HEX signal of Ct < 36. 1 2 x 10 3 RNA copies/µl 2 Threshold cycle (Ct): cycle at which the amplification plot crosses the threshold, i.e., at which there is the first clearly detectable increase in fluorescence. Interpretation of Results A. A FAM fluorescence signal is measured: Positive result, the sample contains BVDV. In such case, a HEX fluorescence signal may be dispensable, because very high concentrations of BVDV RNA may compete with the internal control giving a reduced HEX signal or no HEX signal at all. If the Ct-value of the FAM fluorescence is less than Ct 30 (Ct < 30) the sample is most likely from a persistently infected animal (PI-animal).
- 7 B. A HEX fluorescence signal (internal control) but no FAM fluorescence signal is measured: Negative result, the sample does not contain BVDV. The internal control is measured through HEX fluorescence; complete PCR inhibition is thereby excluded. C. Neither FAM fluorescence nor HEX fluorescence is detected: The test is not valid, no diagnosis possible. As the PCR was inhibited, we recommend to retest the respective individual samples diluted in nuclease free water (e.g. diluted 1:5), to repeat RNA extraction or to repeat the whole procedure starting with new sample material.
- 8 Notes