virotype BTV pan/8 RT-PCR Kit
Detection of all 27 known bluetongue virus serotypes, plus separate detection of the European BTV serotype 8 (BTV-8)
The virotype BTV pan/8 RT-PCR Kit allows the safe detection of RNA from bluetongue virus (BTV) in samples from cattle, sheep, and goats. The kit contains all of the reagents for multiplex real-time RT-PCR in a single, ready-to-use reaction mix. It can be used with pools of up to 10 individual samples of blood and with tissue samples from the spleen and lymph nodes.
The virotype BTV pan/8 RT-PCR Kit is for veterinary use only. In Germany: Licensed in accordance with § 17c of the German Law on Animal Diseases. MA No.: FLI-B 539 In Switzerland: Licensed by the Federal Food Safety and Veterinary Office (FSVO)
Performance
A validation study of 338 blood samples, with known BTV status (174 positive and 164 negative; kindly provided by state laboratories), was tested with the virotype BTV pan/8 RT-PCR Kit. In this study, all positive samples tested positive and all negative samples tested negative, providing a diagnostic sensitivity and specificity of 100%. In addition, a titration series was performed with ten-fold dilutions from 107 to 1 copy/well of BTV RNA. The results demonstrate a high correlation (99%) between the number of copies and the amount of amplification product in the range from 107 to 100 copies/well, and positive detection of the virus down to 1 copy/well (see the figure Sensitive detection of BTV RNA). A comparative study of the virotype BTV pan/8 and the Pan-BTV-S5 RT-PCR (established in-house National Reference Laboratory [NRL] method) was carried out using RNA samples of 24 BTV serotypes (RNA kindly provided by the NRL, Friedrich-Loeffler-Institute). The virotype BTV pan/8 demonstrated a greater sensitivity for detection of BTV (see the table Detection of 24 BTV serotypes).
Detection of 24 BTV serotypes
In this study, RNA samples of 24 BTV serotypes were tested. A direct comparison between the virotype BTV pan/8 RT-PCR Kit and the in-house Pan-BTV-S5 RT-PCR method was carried out. The data illustrates both the detection of all 24 BTV serotypes and the relative greater sensitivity with the virotype BTV pan/8 RT-PCR Kit.
Principle
This is a real-time RT-PCR kit for the detection of RNA from bluetongue virus, the causative agent of the highly infectious bluetongue disease, which mainly affects cattle, sheep, and goats. RT-PCR is based on the amplification of specific regions of the pathogen genome. It is a sensitive and specific tool for pathogen detection. Real-time RT-PCR involves the reverse transcription of viral RNA and subsequent amplification of the cDNA by 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 BTV pan/8 RT-PCR Kit uses three specific primer/probe combinations: one for the RNA of the 24 BTV serotypes known at time of development, one for BTV-8 RNA, and one for the control RNA.
Procedure
The kit contains a ready-to-use PCR mix and an enzyme mix with all of the necessary reagents for the detection of BTV RNA and the required positive and negative controls. With this kit, both reverse transcription and PCR are performed in one reaction tube, reducing the risk of contamination.
RNA is extracted from the sample material (blood or tissue) using a suitable sample preparation kit. The extracted RNA is used for the RT-PCR, which is run on a thermal cycler.
Applications
The virotype BTV pan/8 RT-PCR Kit is for detection of RNA from bluetongue virus (BTV) in samples of from cattle, sheep, and goats. |
