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virotype BTV pan/4 RT-PCR Kit

Detection of RNA from all 27 known bluetongue virus serotypes plus separate identification of BTV serotype 4 (BTV-4) in a single PCR run
  • Separate detection of BTV serotype 4 (BTV-4)
  • Easy-to-use single reaction mix containing controls
  • RT-PCR run time of less than 2 hours
  • Can be performed in one cycler run with other virotype RT-PCR assays

 

The virotype BTV pan/4 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.
Cat No./ID: 280443
virotype BTV pan/8 RT-PCR Kit (24)
For 24 reactions: Master Mix, Positive Control, Negative Control
Cat No./ID: 280445
virotype BTV pan/8 RT-PCR Kit (96)
For 96 reactions: Master Mix, Positive Control, Negative Control
Cat No./ID: 280453
virotype BTV pan/4 RT-PCR Kit (24)
For 24 reactions: Master Mix, Positive Control, Negative Control
Cat No./ID: 280455
virotype BTV pan/4 RT-PCR Kit (96)
For 96 reactions: Master Mix, Positive Control, Negative Control
The virotype BTV pan/4 RT-PCR Kit is for veterinary use only. 
In Germany: Licensed in accordance with § 11 (2) of the German Animal Health Act. MA No.: FLI-C 020
Performance
The virotype BTV pan/4 RT-PCR Kit detects RNA from all 27 known serotypes of bluetongue virus.
Analytical sensitivity was determined by a titration series of in vitro BTV RNA, resulting in the detection of 1–10 RNA copies in the BTV pan and the BTV-4 system. The virotype BTV pan/4 RT-PCR Kit specifically detects BTV-4 (Cy5) and at least 27 BTV serotypes (FAM). BTV-26 is detected by virotype BTV pan/4 RT-PCR Kit using in silico PCR. (See the table "Testing of the BTV serotypes 1–25 and 27 using the virotype BTV pan/4 RT-PCR Kit".)

Testing of the BTV serotypes 1–25 and 27 using the virotype BTV pan/4 RT-PCR Kit
BTV serotype
CT BTV pan (FAM) CT BTV-4 (Cy5)
1 29.39
2 27.49
3
26.05
4 29.21 29.30
5 26.05
6 29.14
7 26.04
8 30.32
9 26.32
10 27.17
11 27.33
12 25.91
13 26.16
14 26.74
15
27.28
16
27.87
17 28.44
18 27.56
19 27.13
20 27.21
21 26.96
22
26.87
23 27.29
24 27.42
25 28.88
27 22.97

For validation of the virotype BTV pan/4 RT-PCR Kit a total of 482 samples were tested. 138 of 482 samples were BTV-4–positive, 119 BTV-8–positive, 6 BTV-5–positive and 202 BTV-negative. BTV-4–positive samples were kindly provided by various eastern and southern European diagnostic laboratories. Additionally, BTV-positive and negative samples were tested in a European field validation study. (See the table "Testing of BTV-4 positive blood samples using the virotype BTV pan/4 RT-PCR Kit and a reference test".)
Testing of BTV-4 positive samples using the virotype BTV pan/4 RT-PCR Kit (only a subset of validation data are displayed)
     virotype BTV pan/4 RT-PCR
 Origin of sample/BTV-4 outbreak  Dilution
 CT BTV pan  CT BTV-4
11/2015 Slovenia
 1:100  32.52  32.95
2014–15 Hungary
 1:10  26.72  28.05
2014–15 Czech Republic
 1:100  30.01  31.40
2014/2015 Bulgaria              –  20.84  21.55
09/2014 Serbia 
 1:10  21.18  23.43
2013 Croatia 
 –  28.98  31.35
2004 Spain 
 1:100  20.36  20.93
1999 Greece 
 1:10  9.99  9.39
Bulgaria
 1:104  23.98  24.78
Morocco  1:104  24.70  25.22

The validation results show that BTV-4 was specifically identified. The virotype BTV pan/4 RT-PCR Kit demonstrated diagnostic sensitivity and specificity of 100%. In addition, no cross-reactivity with other ruminant pathogens tested was detected.
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/4 RT-PCR Kit uses three specific primer/probe combinations: one for the RNA of at least 27 known BTV serotypes, one for BTV-4 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/4 RT-PCR Kit is for detection of RNA from bluetongue virus (BTV) in samples of from cattle, sheep, and goats.