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virotype PRRSV RT-PCR Kit

For detection of RNA from PRRSV in samples from swine

  • Simultaneous detection of the NA and EU genotypes of PRRSV
  • Differentiation of the highly pathogenic PRRSV HP strain
  • Easy-to-use single reaction mix containing controls
  • RT-PCR run time of less than 2 hours



The virotype PRRSV RT-PCR Kit allows the safe and simultaneous detection of the North American (NA) and European (EU) genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) in samples from swine. It can also differentiate the HP strain of the NA genotype. 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 5 individual samples of blood, serum, tissue, bronchial swabs, bronchial lavage, saliva, and semen samples from swine and cell culture supernatant and collective samples of saliva.

The virotype PRRSV RT-PCR Kit is intended for laboratory use. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. The listed testing kits, reagents, and methods are approved by the Friedrich-Loeffler-Institute in Germany for veterinary diagnostic use.

In Germany: Registered in accordance with §17c of the German Law on Animal Diseases. Registration No.: FLI-B 562
Cat No./ID: 282303
virotype PRRSV RT-PCR Kit (24)
For 24 reactions: PRRSV Mix, Positive Control, Negative Control
Cat No./ID: 282305
virotype PRRSV RT-PCR Kit (96)
For 96 reactions: PRRSV Mix, Positive Control, Negative Control

The virotype PRRSV RT-PCR Kit is intended for laboratory use. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. The listed testing kits, reagents, and methods are approved by the Friedrich-Loeffler-Institute in Germany for veterinary diagnostic use.


In Germany: Registered in accordance with §17c of the German Law on Animal Diseases. Registration No.: FLI-B 562
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Sensitive detection of PRRSV.

Using the virotype PRRSV RT-PCR Kit, real-time RT-PCR was performed in triplicate on a titration series of samples of PRRSV EU1, EU2, NA, and HP in ten-fold dilutions from 108 to 10 copies/well.

Performance
The virotype PRRSV RT-PCR Kit detects RNA from porcine reproductive and respiratory syndrome virus. A validation study of 384 samples, with known PRRSV status (129 positive and 255 negative; samples provided by the Friedrich-Loeffler-Institute, state, and private laboratories), was tested with the virotype PRRSV RT-PCR Kit. Of 129 positive samples, 123 samples tested positive, providing a diagnostic sensitivity of > 95%. In this study all negative samples tested negative, providing a diagnostic specificity of 100%. In addition, a titration series was performed with ten-fold dilutions from 108 to 10 copies/well of PRRSV RNA. The results demonstrate a high correlation (99%) between the number of copies and the amount of amplification product in the range from 108 to 100 copies/well, and positive detection of the virus down to 10 copies/well (see the figure "Sensitive detection of PRRSV"). The analytical sensitivity of the kit was compared to that for two other commercially available kits. An RNA panel of 12 PRRSV reference strains (kindly provided by Dr. Bernd Hoffmann, Friedrich-Loeffler-Institute) was tested with all three kits. The virotype PRRSV RT-PCR Kit gave comparable results to the kit from Supplier A and was more sensitive than the kit from Supplier B (see the table Comparison of analytical sensitivity). Only the virotype PRRSV RT-PCR Kit could detect the highly pathogenic (HP) strain of the NA genotype.

Comparison of analytical sensitivity
Kit   virotype PRRSV RT-PCR Kit Kit from
Supplier A 
Kit from Supplier B 
Target  EU  NA  HP  IC  EU  NA  IC  EU  NA  IC 
EU strains                     
EU Stendal V953  25.22       34.61  24.08    29.89  24.95    35.66 
Intervet  22.02      33.22  19.41    28.85  24.65     
Cobbelsdorf  23.86       32.65  22.20    29.32  26.02     
Stendal V852  23.24       39.96  21.86    29.68  26.16    36.98 
Stendal V1904  24.30      39.06  23.47    29.30  27.63    37.41 
Stendal V1952/97  26.29      35.41  24.51    31.32  28.12    36.06 
                     
NA strains                     
Stendal V1445/99    21.39    34.85    21.17  30.78    24.92  34.10 
USA VD28775/2    21.35    29.14    21.61  30.40    24.07  39.08 
USA VD29949/17    22.41    28.48    22.18  29.65    26.89   
USA 2    19.83    30.64    20.44  30.81    23.64   
USA 18    19.06    31.48    19.50  30.39    26.54   
China    20.87  22.44  28.36    20.54  29.89    25.84   
                     
Mean Value EU  24.16        22.59      26.26     
Mean Value NA    20.82        20.91      25.32   

Using the virotype PRRSV RT-PCR Kit and two other commercially available PRRSV detection kits, real-time RT-PCR was performed in triplicate on an RNA panel of 12 reference strains of the virus. The average CT values for the targets are shown.
Principle

This is a real-time RT-PCR kit for the detection of RNA from porcine reproductive and respiratory syndrome virus. 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.

Procedure

The kit contains a ready-to-use mix with all of the necessary reagents for the detection of PRRSV 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, serum, tissue, saliva, semen, bronchial swabs or lavage, or cell culture supernatants) using a suitable sample preparation kit. The extracted RNA is used for the RT-PCR, which is run on a thermal cycler.

The kit uses four specific primer/probe combinations. Each is detected in a different fluorescence channel, allowing the simultaneous detection of the different PRRSV strains.

Applications

The virotype PRRSV RT-PCR Kit is for the detection of porcine reproductive and respiratory syndrome virus in samples of blood, serum, tissue, saliva, semen, bronchial swabs or lavage, and cell culture supernatants from swine.

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