Lsi vetmax prrsv eu na real time pcr kit

In order to correlate IDV with respiratory disease and to exclude other causative agents, clinical specimens from IDV positive herds were also subjected to standard bacteriological and/or virological PCR tests against the most common swine respiratory pathogens. These included: Actinobacillus pleuropneumoniae (APP)²⁶ , swine IAV^{27 (link)}, Porcine respiratory reproductive (PRRS) (LSI VetMAX™ PRRSV EU/NA Real-Time PCR Kit, Thermo Fisher Scientific, Waltham, MA USA) virus and Porcine Circovirus 2 virus (PCV-2)^{28 (link)}.

All SDPP samples were re-solubilized in distilled water at the ratio 1:9 of SDPP: water volume to represent the typical solid content in liquid plasma. Two hundred milliliters of diluted plasma sample were used for nucleic acid extraction using MagMAX^™ Pathogen RNA/DNA Kit (Thermo Fisher Scientific, MA, USA). The recommended quantity of purified nucleic acids was amplified using real time PCR kits for PCV-2 (LSI VetMAX^™ Porcine Circovirus Type 2 Quantification, Thermo Fisher Scientific, MA, USA), Porcine reproductive and respiratory syndrome virus [PRRSV] European and North American strains (LSI VetMAX^™ PRRSV EU/NA Real-Time PCR Kit; Thermo Fisher Scientific, MA, USA), Swine influenza virus [SIV] (EXOone Influenza A, EXOPOL, Zaragoza, Spain), Porcine parvovirus [PPV] (VetMAX^™ Porcine Parvovirus Kit, Thermo Fisher Scientific, MA, USA), PEDV, Transmissible gastroenteritis virus [TGEV] and Swine deltacoronavirus [SDCoV] (VetMAX^™ PEDV/TGEV/SDCoV, Thermo Fisher Scientific, MA, USA) and Senecavirus A [SVA] (EXOone Seneca Virus Valley, EXOPOL, Zaragoza, Spain).
According to all PCR kit guidelines, virus genome results with Ct values >40 were considered negative.

Viral RNA was extracted from 200 μl of sample (serum or lung homogenate) using a commercial kit (NucleoMag® Vet kit, Macherey-Nagel, Düren, Germany), according to the manufacturer's instructions. An exogenous internal control RNA (Xeno^TM RNA control, Applied Biosystem) was added to specimens prior to RNA extraction to verify the success of the procedure and the absence of inhibitors. The extraction was carried out on the Biosprint 96 instrument (Qiagen, Hilden, Germany), using the NucleoMag Vet 200 protocol. Nucleic acids were eluted into 100 μl of elution buffer and immediately subjected to RT-PCR or stored at −80°C until used. RNA was detected using the LSI VetMAX^(TM) PRRSV EU/NA Real-Time PCR kit (Applied Biosystem), as specified by the manufacturer.
Ten-fold serial dilutions in nuclease-free water of an EU PRRSV RNA (3.5 × 10⁵ to 35 copies/μl) were used to generate a standard curve and to quantify PRRSV RNA in the samples. Triplicates of each dilution were run in each assay. The following equation: $\text{x}=10\frac{\text{Cq}-\left(\text{yintercept}\right)}{\text{slope}},$
where x represents the genome copies per microliter and was used to transform the Cq values of the samples into estimates of genome copies of PRRSV RNA per milliliter.
The detection limit of PRRSV-1 target was nine copies of nucleic acids per reaction, corresponding to 0.642 genome copies/μl.