Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is an RNA virus of the Arterivitridae family which is the causative agent of Porcine Reproductive and Respiratory Syndrome, a fatal disease that causes significant economic losses in the pig industry. The single-stranded, positive-sense RNA genome is 15,428 nucleotides long and comprises atleast eight open reading frames (ORFs) that encode about 20 proteins. The ORFs 1a and 1b comprise about 80 per cent of the genome and encode an RNA-dependent RNA polymerase called RNA replicase. The six smaller ORFs, 2 to 7, located at the 3’ end of the genome encode a number of viral structural proteins associated with the virion, including the envelope protein (E) and nucleocapsid protein (N). There are currently two known strains of PRRSV –Northern American strain (VR-2332) and European strain (Leystad Virus), which are only 55–70 per cent identical at the nucleotide level. Transmission of the virus can occur by several routes: inhalation, ingestion, exposure by artificial insemination or by parenteral exposure. It is thought that the virus targets the alveolar lung macrophages as well as macrophages of other tissues and testicular germ cells, enteringthe host cells by the standard endocytotic pathway in clathrin-coated pits. Within macrophages the viral replication machinery can be found within double-membrane vesicles formed by the host cells endoplasmic reticulum. After replication, new nucleocapsids assemble and bud into the lumen of the ER where they accumulate in vesicles and move to the plasma membrane where fusion takes place resulting in viral release
Infection with this virus can cause severe reproductive damage resulting in premature farrowing, stillborn piglets and weak piglets which die soon after birth. Clinical signs of the infection include fever, lethargy, pneumonia, anorexia, and discoloration of the ears and vulva. Infected pigs persistently shed virus via blood, saliva, milk and colostrums, urine and feces.
Specificity:The Primerdesign genesig Kit for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) genomes is designed for the in vitro quantification of PRRSV genomes. The kit is designed to have the broadest detection profile possible whilst remaining specific to the PRRSV genome
The primers and probe sequences in this kit have 100% homology with a broad range of PRRSV sequences based on a comprehensive bioinformatics analysis.
Kit Contents:
US specific primer/probe mix (BROWN) Once resuspended the kits should remain at -20ºC until ready to use.
EU specific primer/probe mix (BROWN) Once resuspended the kits should remain at -20ºC until ready to use.
Test Principle:There are currently two known subtypes of PRRSV – Northern American strain (VR-2332) and European strain (Leystad Virus), which are only 55–70 per cent identical at the nucleotide level. The kit has been developed with two primer and probe sets in order to ensure thedetection of both the subtypes and to differentiate between them.
Positive Control:For copy number determination and as a positive control for the PCR set up, the kit contains a positive control template. This can be used to generate a standard curve of PRRSV copy number / Cq value. Alternatively the positive control can be used at a single dilution where fullquantitative analysis of the samples is not required. Each time the kit is used, at least one positive control reaction must be included in the run. A positive result indicates that the primers and probes for detecting the target PRRSV gene worked properly in that particular experimental scenario. If a negative result is obtained the test results are invalid and must berepeated. Care should be taken to ensure that the positive control does not contaminate any other kit component which would lead to false-positive results. This can be achieved by handling this component in a Post PCR environment. Care should also be taken to avoid cross-contamination of other samples when adding the positive control to the run. This can be avoided by sealing all other samples and negative controls before pipetting the positive control into the positive control well.
Negative Control:To validate any positive findings a negative control reaction should be included every time the kit is used. For this reaction the RNase/DNase free water should be used instead of template. A negative result indicates that the reagents have not become contaminated while setting up the run.
Suitable Sample Material:All kinds of sample material suited for PCR amplification can be used. Please ensure the samples are suitable in terms of purity, concentration, and DNA integrity. Always run at least one negative control with the samples. To prepare a negative-control, replace the template DNA sample with RNase/DNase free water.
Dynamic Range of Test:Under optimal PCR conditions genesig PRRSV detection kits have very high priming efficiencies of >95% and can detect less than 100 copies of target template.
KIT STORAGE AND STABILITY:This kit is stable at room temperature but should be stored at --20°C on arrival. Once the lyophilised components have been resuspended they should not be exposed to temperatures above -20°C for longer than 30 minutes at a time and unnecessary repeated freeze/thawing should be avoided. The kit is stable for six months from the date of resuspension under these circumstances
If a standard curve dilution series is prepared this can be stored frozen for an extended period. If you see any degradation in this serial dilution a fresh standard curve can be prepared from the positive control
We do not recommend using the kit after the expiry date stated on the pack.
